Coding sequence for protein phosphatase methylesterase, recombinant DNA molecules and methods

ABSTRACT

Carboxymethylation of proteins is a highly conserved means of regulation in eukaryotic cells. The protein phosphatase 2A (PP2A) catalytic (C) subunit is reversibly methylated at its carboxy-terminus by specific methylesterase. Carboxymethylation affects PP2A activity and varies during the cell cycle. The present disclosure provides the coding sequence of a methylesterase, herein named Protein Phosphatase Methylesterase-1 (PME-1). PME-1 is highly conserved from yeast to human and contains a motif found in lipases, which motif has a catalytic triad-activated serine as the active site nucleophile. Recombinant PME-1 polypeptide produced in bacteria demethylates PP2A C subunit in vitro and okadaic acid, a known inhibitor of the PP2A methylesterase, inhibited this reaction. PME-1 represents the first mammalian protein phosphatase methylesterase cloned to date.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional application of U.S. applicationSer. No. 09/839,497 filed Apr. 20, 2001, which is a divisionalapplication of U.S. application Ser. No. 09/293,322 filed Apr. 16, 1999and claims priority from U.S. Provisional Application Serial No.60/082,202, filed Apr. 17, 1998.

ACKNOWLEDGMENT OF FEDERAL RESEARCH SUPPORT

[0002] This invention was made, at least in part, with funding from theUnited States National Institutes of Health (Grant CA 57327).Accordingly, the United States Government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

[0003] The field of this invention is the area of molecular biology, andin particular the DNA sequence encoding Protein PhosphataseMethylesterase-1 (PME-1, formerly called p44A), recombinant vectors, andmethods for recombinant production of PME-1 demethylase and its use inidentifying compositions with inhibitory activity.

[0004] Protein phosphatase 2A (PP2A) is a highly conservedserine/threonine phosphatase involved in the regulation of a widevariety of enzymes, signal transduction pathways, and cellular events[Cohen, P. (1989) Annu. Rev. Biochem. 58:453-508; Lee, T. H., et al.(1991) Cell 64:415-423; Mayer-Jaekel, R. E. et al. (1993) Cell72:621-633; Sontag, E. S. et al. (1993) Cell 75:887-897; Uemura, T. etal. (1993) Genes Dev. 7:429-440]. The minimal structure thought to existin vivo consists of a heterodimer between a catalytic 36 kDa subunittermed C and a constant regulatory 63 kDa subunit termed A [Kremmer, E.et al. (1997) Mol. Cell Biol. 17:1692-1701; Usui, H. et al. (1988) J.Biol. Chem. 263:3752-3761]. This heterodimer is often further complexedwith one of several additional regulatory subunits termed B, B′, and B″[Cohen, P. (1989) supra]. In PP2A heterotrimers, the A subunit binds toboth the catalytic C and regulatory B-type subunits [Ruediger, R. et al.(1992) J. Virol. 68:123-129; Ruediger, R. et al. (1994) Mol. Cell Biol.12:4872-4882]. In the case of the B subunit, it has been shown that oneor more of the nine C subunit carboxy terminal amino acids are essentialfor heterotrimer formation [Ogris, E. et al. (1997) Oncogene15:911-917]. In cells stably transformed by the middle tumor antigen(MT) of polyomavirus, MT is found in place of the B subunit in a smallportion (−10%) [Ulug, et al. (1992) J. Virol. 66:1458-1467] of PP2Acomplexes [Pallas, D. C. et al. (1990) Cell 60:167-176]. MT/PP2A complexformation is important for MT-mediated transformation [Grussenmeyer, etal. (1987) J. Virol. 61:3902-3909; Pallas, et al. (1988) J. Virol.62:3934-3940; Glenn, G. M. et al. (1995) J. Virol. 69:3729-3736;Campbell, K. S. et al. (1995)J Virol. 69:3721-3728]. Unlike for Bsubunit, formation of PP2A heterotrimers containing MT does not requirethe last nine amino acid residues of the C subunit [Ogris, E. et al.(1997) supra]. The small tumor antigens (STs) of various papovavirusesalso form complexes with the A and C subunits of PP2A [Pallas, D. C. etal. (1990) supra].

[0005] Consistent with the multiple important roles that PP2A plays indiverse pathways and cellular events, PP2A is highly regulated. Theregulatory mechanisms include modulation by regulatory subunits orinhibitory proteins and modulation by post-translational modification ofthe C subunit. Subunit composition of the PP2A complex affects bothcatalytic activity and substrate specificity [Agostinis, P. et al.(1992) Eur. J. Biochem. 205:241-248; Favre, B. et al. (1994) J. Biol.Chem. 269:16311-16317; Scheidtmann, K. H. et al. (1991) Mol. Cell. Biol.11:1996-2003; Sola, M. M. et al. (1991) Biochem. Biophys. Acta1094:211-216]. In the case of B subunit, changes of up to 100 fold havebeen documented using cdc2 phosphorylated substrates [Agostinis, P. etal. (1992) Eur. J. Biochem. 205:241-248; Ferrigno, P. et al. (1993) Mol.Biol. Cell 4:669-677; Mayer-Jaekel, R. E. et al. (1994) Journal of CellScience 107:2609-2618; Ogris, E. et al. (1997) supra; Sola, M. M. et al.(1991) Biochem. Biophys. Acta 1094:211-216]. Two PP2A inhibitor proteinshave been reported: I1PP2A (also called PHAPI) and I2PP2A (also calledPHAPII or SET) [Li, M. et al. (1996) Biochemistry 34:1988-1996; Li, M.et al. (1996) Biochemistry 35: 6998-7002; Li, M. et al. (1995) J. Biol.Chem. 271:11059-11062]. These also appear to be substrate-dependent intheir effects. Perusal of the NCBI GenBank and EST databases via BLASTfollowed by sequence comparisons using DNASTAR MegAlign softwareindicates the existence of three different human PHAPI isoforms encodedby different genes and the presence of multiple alternatively splicedforms of PHAPII. A Xenopus homolog of PHAPII was recently shown tointeract with B-type cyclins in vitro [Kellogg, D. R. et al. (1995) J.Cell Biol. 130:661-673], but the molecular consequences of thisinteraction in the regulation of PP2A are not known.

[0006] The post-translational modifications of the C subunit that havebeen reported to modulate PP2A activity include phosphorylation andmethylation. Inhibition of PP2A activity in vitro was found upon Csubunit phosphorylation at either tyrosine 307 or at one or moreunidentified threonine residues [Chen, J. et al. (1992) Science257:1261-1264; Guo, H. and Damuni, Z. (1993) Proc. Natl. Acad. Sci. USA90:2500-2504]. A similar modification may occur in vivo in response totransformation or growth stimulation [Chen, J. et al. (1994) J. Biol.Chem. 269:7957-7962]. The first indication that PP2A C subunit wasmethylated involved two observations. A 36 kDa SV40 small tumor antigen(ST)-associated cellular protein is a major acceptor of the methyl groupfrom radiolabeled S-adenosyl methionine added to cell extracts [Rundell,K (1987) J. Virol. 61:1240-1243]. This ST-associated cellular proteinwas reported to be the PP2A C subunit [Pallas, D. C. et al. (1990)supra]. The site of methylation of the PP2A C subunit has beenidentified as leucine 309 [Favre, B. et al. (1994) supra; Lee, J. andStock, J. (1993) J. Biol. Chem. 268:19192-19195; Xie, H. and Clarke, S.(1994) J. Biol. Chem. 269:1981-1984]. One study reported anapproximately two-fold increase in the activity of PP2A uponmethylation, adjusting for the stoichiometry of methylation [Favre, B.et al. (1994) supra]. Only phosphorylase a and the peptide substrate,phosphorylated Kemptide, were used in that study. These substrates oftengive similar results. Thus, it remains to be determined whether greatereffects might be observed with other substrates. Based on differentialantibody recognition of methylated and non-methylated C subunit, PP2Ahas been reported to undergo cell cycle dependent changes in methylation[Turowski, P. et al. (1995) J. Cell Biol. 129:397-410]. It is not knownwhether methylation of PP2A affects the subunit composition of theenzyme. Partially purified fractions of PP2A containing A/C heterodimersor A/B/C heterotrimers have both been shown to be substrates for thePP2A methyltransferase [Xie, H. and Clarke, S. (1994) supra]. There arealso data which indicate that methylated C subunit can associate withSV40 ST [Rundell, K. (1987) supra].

[0007] The B subunit functions in cell cycle progression through mitosisand in cytokinesis [Healy, A. M. et al. (1991) Mol. Cell Biol.11:5767-5780; Mayer-Jaekel, R. E. et al. (1993) supra; Uemura, T. et al.(1993) Genes Dev. 7:429-440]. In cells stably transformed by the middletumor antigen (MT) of polyomavirus, MT is found in place of the Bsubunit in a small portion (˜10%) [Ulug, E. T. et al. supra] of PP2Acomplexes [Pallas, D. C. et al. (1990) supra]. MT/PP2A complex formationis known to be important for MT-mediated transformation [Campbell, K. S.et al. (1995) supra; Glenn, G. M. et al. (1995) supra; Grussenmeyer, T.et al. (1987) supra; Pallas, D. C. et al. (1988) supra], but the precisefunctional consequences of MT association with PP2A are still beingelucidated. It was recently shown that there is a requirement for directB/C subunit interaction to form stable heterotrimers [Ogris, E. et al.(1997) supra].

[0008] The nine carboxy-terminal amino acids of the PP2A C subunit,residues 301 to 309, include tyrosine 307, the site of phosphorylationin vitro by v-src, and two potential sites of threonine phosphorylation,residues 301 and 304. Seven of these nine residues, including threonine304 and tyrosine 307, are found in every PP2A C subunit cloned to date.Threonine 301 is somewhat less conserved.

[0009] In order to study cellular proteins which interact with PP2A, twocatalytically inactive C subunit mutants were generated and used to formstable complexes. The present invention describes the identification ofone of these proteins, herein named Protein Phosphatase Methylesterase-1(PME-1).

[0010] Due to the fact that PP2A is shown to regulate multiple cellularpathways by dephosphorylating several key proteins, there has been along felt need in the art to understand the molecular mechanisms bywhich PP2A activity is modulated. The present invention describescloning of one such modulating enzyme for human PP2A, named hereinPME-1, and also shows how to produce recombinant PME-1 polypeptide,which is then used in in vitro assays to identify inhibitors for PME-1activity.

SUMMARY OF THE INVENTION

[0011] It is an object of the present invention to provide nucleotidesequences encoding protein phosphatase methylesterase-1 (PME-1) and thededuced amino acid sequence therefor. Specifically exemplified codingsequences are given in Table 2, together with the deduced amino acidsequence for the human; Tables 6 and 3 for the yeast; Tables 7 and 4 forthe nematode. All synonymous coding sequences for the exemplified aminoacid sequences are within the scope of the present invention.

[0012] It is a further object of the present invention to providefunctionally equivalent coding and protein sequences, includingequivalent sequences from other mammals and other organisms, includingbut not limited to yeast and nematodes, and variant sequences fromhumans. Functionally equivalent PME-1 coding sequences are desirablyfrom about 50% to about 80% nucleotide sequence homology (identity) tothe specifically identified PME-1 coding sequence, from about 80% toabout 95%, and desirably from about 95% to about 100% identical incoding sequence to the specifically exemplified coding sequence. Eachinteger and each subset of each specified range is intended within thecontext of the present invention.

[0013] Hybridization conditions of particular stringency provide for theidentification of homologs of the human PME-1 coding sequence from otherspecies and the identification of variant human sequences, where thosehomologs and/or variant sequences have at least (inclusively) 50 to 85%,85 to 100% nucleotide sequence identity, 90 to 100%, or 95 to 100%nucleotide sequence identity.

[0014] The PME-1 coding sequence and methods of the present inventioninclude the homologous coding sequences in organisms other than humansand mice. Methods can be employed to isolate the corresponding codingsequences (for example, from cDNA) from other organisms, including butnot limited to other mammals, avian species, Saccharomyces andCaenorhabditis elegans useful in the methods of this invention using thesequences disclosed herein and experimental techniques well known to theart.

[0015] It will further be understood by those skilled in the art thatother nucleic acid sequences besides those disclosed herein for thePME-1 coding sequence will function as coding sequences synonymous withthe exemplified coding sequences. Nucleic acid sequences are synonymousif the amino acid sequences encoded by those nucleic acid sequences arethe same. The degeneracy of the genetic code is well known to the art.For many amino acids, there is more than one nucleotide triplet whichserves as the codon for a particular amino acid, and one of ordinaryskill in the art understands nucleotide or codon substitutions which donot affect the amino acid(s) encoded.

[0016] Specifically included in this invention are PME-1 sequences fromother organisms than those exemplified herein, which sequences hybridizeto the PME-1 sequence disclosed under stringent conditions. Stringentconditions refer to conditions understood in the art for a given probelength and nucleotide composition and capable of hybridizing understringent conditions means annealing to a subject nucleotide sequence,or its complementary strand, under standard conditions (i.e., hightemperature and/or low salt content) which tend to disfavor annealing ofunrelated sequences. As specifically exemplified, “conditions of highstringency” means hybridization and wash conditions of 65°-68° C.,0.1×SSC and 0.1% SDS (indicating about 95-100% nucleotide sequenceidentity/similarity). Hybridization assays and conditions are furtherdescribed in Sambrook et al. (1989) Molecular Cloning, Second Edition,Cold Spring Harbor Laboratory, Plainview, N.Y.

[0017] As used herein, conditions of moderate (medium) stringency arethose with hybridization and wash conditions if 50-65° C., 1×SSC and0.1% SDS (where a positive hybridization result reflects about 80-95%nucleotide sequence identity). Conditions of low stringency aretypically those with hybridization and wash conditions of 40-50° C.,6×SSC and 0.1% SDS (reflecting about 50-80% nucleotide sequenceidentity).

[0018] As used herein, all or part of a nucleotide sequence refersspecifically to all continuous nucleotides of a nucleotide sequence, ore.g. 1000 continuous nucleotides, 500 continuous nucleotides, 100continuous nucleotides, 25 continuous nucleotides, and 15 continuousnucleotides.

[0019] Where PME-1-homologous coding sequences are to be isolated fromother organisms, one desirably uses nucleotide probes or primers fromthe most highly conserved regions of the PME-1 protein. For example, theskilled artisan desirably uses hybridization probes or PCR primersencoding the active site region (GHSMGGA, amino acids 154-160, SEQ IDNO:5, in the protein sequence) and a second highly conserved sequencewithin the protein [GQMQGK, amino acids 333-338, SEQ ID NO:5) to deriveprobe or primer sequences.

[0020] It is well-known in the biological arts that certain amino acidsubstitutions may be made in protein sequences without affecting thefunction of the protein. Generally, conservative amino acidsubstitutions or substitutions of similar amino acids are toleratedwithout affecting protein function. Similar amino acids can be thosethat are similar in size and/or charge properties, for example,aspartate and glutamate, and isoleucine and valine, are both pairs ofsimilar amino acids. Similarity between amino acid pairs has beenassessed in the art in a number of ways. For example, Dayhoff et al.(1978) in Atlas of Protein Sequence and Structure, Volume 5, Supplement3, Chapter 22, pp. 345-352, which is incorporated by reference herein,provides frequency tables for amino acid substitutions which can beemployed as a measure of amino acid similarity. Dayhoff et al.'sfrequency tables are based on comparisons of amino acid sequences forproteins having the same function from a variety of evolutionarilydifferent sources.

[0021] Also within the scope of the present invention are recombinanthost cells and recombinant vectors carrying the PME-1 coding sequencesof the present invention. Desirably, those coding sequences are operablylinked to transcriptional and translational control sequences functionalin the host cell into which the vectors are introduced and maintained.

[0022] Further provided by the present invention are methods for therecombinant production of a PME-1 protein. After a suitable vector inwhich a PME-1 coding sequence is operably linked to transcriptional andtranslational control sequences is introduced into a recombinant hostcell of choice, the recombinant host cells are cultured under conditionswhere the PME-1 sequences are expressed. The PME-1 can then berecovered, if desired. It is understood that the vector and host cellsare chosen for maintenance of the vector within the host cell.Similarly, the transcriptional and translational control sequences arechosen for function in the host cell of choice. The specificallyexemplified human PME-1 sequence can be modified, for example, usingpolymerase chain reaction (PCR) technology by substituting synonymouscodons according to the known codon usage of the chosen host cell sothat expression of the coding sequence is maximized.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1 shows that the catalytically inactive mutants of PP2A canform complexes with the regulatory A subunit and MT in vivo. Lysatesfrom cells containing only control vector (GRE only) or HA-tagged wt(wt-36) or mutant C subunits (H59Q, H118Q) were precipitated withanti-HA tag antibody (12CA5) and analyzed by SDS-PAGE andimmunoblotting. The blot was probed first with anti-MT antibody, andthen sequentially with antibodies recognizing the A, C (via the epitopetag), and B PP2A subunits. Because a lower level of expression wasconsistently seen with H118Q, the immunoprecipitate of this mutant wasprepared from more cells; to properly control for this, the controlimmunoprecipitate was prepared from an equivalent amount of cellsexpressing only the vector. Under these conditions, a small amount of MTbinds non-specifically to the immunoprecipitate in the GRE only lane.

[0024]FIG. 2A illustrates HA tag immunoprecipitates prepared from³⁵S-labeled cell lines individually expressing HA-tagged wt (36wt) ormutant C subunits (H59Q, H118Q) or vector only (GRE only) analyzed bySDS-PAGE and autoradiography. Portions of the gel where C subunit, Asubunit, and a novel 44 kDa protein migrate are shown. The C subunitsmigrate as doublets in these gels; whether doublets or a single band areseen varies from gel to gel (compare with FIG. 1). Migration of Csubunit as doublets on SDS-PAGE has been noted previously for bothHA-tagged and endogenous PP2A C subunits [Campbell et al. (1995) supra;Ogris et al. (1997) supra; Turowski et al. (1995) supra] and does notappear to be due to degradation. The panels and lanes shown are from thesame experiment and gel, but the lanes were not all originally adjacent.Even on long exposure, the 44 kDa protein seen in the mutant lanes isnot seen in the wt or control lanes.

[0025]FIG. 2B shows immunoprecipitates identical to those in FIG. 2Aanalyzed by 2D gel electrophoresis. Only the portion of each gelcontaining the relevant proteins is shown. The A, B and C subunits andp44B are indicated by labeled brackets and arrowheads, while thecorresponding positions in panels lacking these proteins are indicatedwith unlabeled brackets or arrowheads. For reference, actin is indicatedin all panels by a small, unlabeled arrow.

[0026]FIG. 2C shows silver-stained 2D gels of HA tag immunoprecipitatesprepared from unlabeled cells expressing vector only (GRE only) or the Csubunit mutant, H118Q. Only the portion of each gel containing therelevant proteins is shown. The A and C subunits, PME-1, and anti-HA tagantibody heavy chain (Ab) are indicated by labeled brackets andarrowheads. Unlabeled arrowheads indicate the corresponding positions inthe GRE only control panel. For reference, actin is indicated in bothpanels by a small, unlabeled arrow. The approximate position that p44Bwould be located on these gels is indicated by the unlabeled brackets.

[0027]FIG. 3A is a schematic of a 2.5 kb human PME-1 cDNA. On the stickdiagram, the positions of the in frame 5′ UTR stop codon (TGA), of thefirst two potential start codons (ATGs), of tandem stop codons (TAGTGA)at the end of the PME-1 ORF, and of the poly A tail (bracket) are shown.The 3′ end of the 3′ UTR, including the position of the poly A tail, wasdeduced by analyzing overlapping PME-1 ESTs; all other regions weredirectly sequenced. The sequence shown extends from the in frame 5′ UTRstop codon (TGA; overlined) to the second possible start ATG (doubleunderlined) (SEQ ID NO:16). The first possible start ATG (underlinedonce in the sequence shown) was identified as the authentic start sitein vivo by making constructs whose transcription/translation products invitro would start with one or the other of these two ATGs. ³⁵S-labeledin vitro transcription/translation product starting at the first ATG,but not the product starting at the second ATG, comigrated precisely on2D gels with PME-1 from HeLa cell lysates.

[0028]FIG. 3B shows that PME-1 mRNA is expressed in different tissues.Total RNA from the indicated mouse organs was separated byelectrophoresis and hybridized with a mouse PME-1 partial cDNA probefrom the 3′ UTR of mouse PME-1. In a separate experiment, the size ofthe PME-1 transcript was calculated to be 2.6±0.2 kB. The lower panelshows the 18S rRNA from the same blot visualized with methylene blue.

[0029]FIG. 4 demonstrates that PME-1 stably associates with H59Q but notwild-type C subunit. HA tag immunoprecipitates prepared from NIH3T3(NIH) or MT-transformed NIH3T3 (NIHMT) cell lines individuallyexpressing HA-tagged wt (wt C sub) or mutant (H59Q) C subunits wereanalyzed by SDS-PAGE and immunoblotting with HA tag antibody and PME-1anti-peptide antibody. The C subunits migrate as tight doublets in thesegels. The panels and lanes shown are from the same experiment and gel,but the lanes were not all originally adjacent. Even on long exposure,the 44 kDa protein seen in the mutant lanes is not seen in the wt lanes.

[0030]FIG. 5 shows that human PME-1 is a PP2A methylesterase.Immunoprecipitated PP2A C subunit was incubated with lysates frombacteria either not expressing PME-1 (control) or expressing PME-1(PME-1), or with purified bacterially-expressed PME-1 (˜5 ng). Okadaicacid (O.A.) or PMSF was added to the reactions to the indicated finalconcentrations. Reactions containing 1.25% DMSO as a control to matchthe level resulting from addition of okadaic acid or PMSF stocksolutions are noted. After incubation, the immunoprecipitated PP2A Csubunits were analyzed by SDS-PAGE. Proteins were transferred tonitrocellulose and the membrane was probed with 4b7(methylation-sensitive Ab), an anti-C subunit antibody that onlyrecognizes unmethylated C subunits. Subsequently, the same membrane wasprobed with Transduction Laboratories, (Lexington, Ky.) anti-PP2A Csubunit antibody (methylation-insensitive Ab), which is insensitive tothe methylation state of PP2A and therefore reveals the total C subunitin each lane. The C subunits migrated as doublets in this gel, butwhether double or single bands are seen can vary (see comments in legendto FIG. 2A).

[0031]FIG. 6A shows that the PP2A inhibitors, okadaic acid, sodiumfluoride, and sodium pyrophosphate, reduce the amount of PME-1 complexedwith the catalytically inactive H59Q C subunit. Seven parallel dishes ofNIH3T3 cells expressing HA-tagged H59Q were lysed in NP40 lysis buffercontaining the indicated inhibitor(s) at the following concentrations:sodium vanadate (1 mM); NaF (50 mM); okadaic acid (500 nM);phenylarsineoxide (PAO; 10 μM); sodium pyrophosphate (Na₄P₂O₇; 20 mM).Anti-HA tag immunoprecipitates were prepared from these lysates andanalyzed by SDS-PAGE and immunoblotting. The blot was probedsequentially with antibodies detecting PME-1 and H59Q C subunit (via itsHA tag). In a separate experiment using phosphorylase a as substrate,sodium fluoride, okadaic acid and sodium pyrophosphate were respectivelyfound to inhibit PP2A 91±10%, 97±4%, and >99%, while phenylarsineoxideand sodium vanadate respectively showed no or 25±18% inhibition.

[0032]FIG. 6B shows that loss of the C subunit carboxy-terminus reduces,but does not abolish, PME-1 Binding. Non-immune (N) and HA tag (I)immunoprecipitates were prepared from MT-transformed NIH3T3 cellsexpressing vector only (GRE only), HA-tagged H59Q, or HA-taggedH59Q/301Stop double mutant which lacks nine carboxy-terminal aminoacids. Immune complexes were analyzed by SDS-PAGE; proteins weretransferred to nitrocellulose; and immunoblotting was performed withantibodies directed against A subunit, PME-1, and C subunit (anti-HAtag). The C subunits migrate as doublets in this gel, but whether doubleor single bands are seen can vary (see comments in legend to FIG. 2A).The band seen in all lanes in the PME-1 panel is from theimmunoprecipitating antibodies. Chemiluminescent quantitation (using aBiorad Fluor-S Max Multilmager, Hercules, Calif.) was used in sevenseparate experiments with mixtures of clones to quantify the ratio ofPME-1 to C subunit signal in each lane. In six of seven experiments withmixes of clones, the double mutant bound less PME-1 than did H59Q, witha mean reduction of 56±30% and a median value of 39 (range of 8-87%).Thus, PME-1 binding is clearly reduced by loss of the carboxy-terminus.In a seventh experiment, for unknown reasons, the double mutant bound235% of the H59Q level of PME-1, lowering the overall mean reduction to28% (median=40).

[0033]FIG. 6C demonstrates that subunit carboxy-terminal antibodiesimmunoprecipitate reduced amounts of H59Q/PME-1 Complex.Immunoprecipitates were prepared from MT-transformed NIH3T3 cellsexpressing HA-tagged H59Q using control antibody, HA-tag antibody(12CA5), or carboxy-terminal C subunit antibodies (1D6, 4B7, 4E1). Theimmune complexes were analyzed by SDS-PAGE; proteins were transferred tonitrocellulose; and immunoblotting was performed with anti-A subunitantibody (upper panel), anti-PME-1 antibody (middle panel) and anti-Csubunit antibody recognizing both endogenous and HA tagged proteins(1D6; lower panel). The positions of A subunit, the immunoprecipitatingantibody heavy chains (Ab), PME-1, HA-tagged H59Q C subunit, anduntagged, endogenous wt C subunit are indicated. The C subunits migrateas single bands in this gel, but whether double or single bands are seencan vary (see comments in legend to FIG. 2A). HA-tagged H59Q C subunitmigrates more slowly than endogenous wt C subunit because of the HA tag.

DETAILED DESCRIPTION OF THE INVENTION

[0034] “Nucleic acids” and “polynucleotides,” as used herein, may be DNAor RNA. One of skill will recognize that the sequences from nematodegenes used in the methods of the invention need not be identical and maybe substantially identical (as defined below) to sequences disclosedhere. In particular, where a polynucleotide sequence is transcribed andtranslated to produce a functional polypeptide, one of skill in the artrecognizes that because of codon degeneracy, a number of synonymouspolynucleotide sequences will encode the same polypeptide. Similarly,because amino acid residues share properties with other residues,conservative substitutions of amino acids within a polypeptide may leadto distinct polypeptides with similar or identical function.

[0035] The term “operably linked” refers to functional linkage, forexample, between a promoter and a downstream sequence, wherein thepromoter sequence initiates transcription of the downstream sequence.

[0036] “Percentage of sequence identity” for polynucleotides andpolypeptides is determined by comparing two optimally aligned sequencesover a comparison window, wherein the portion of the polynucleotide orpolypeptide sequence in the comparison window may comprise additions ordeletions (i.e. gaps) as compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid base or amino acid residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison and multiplying the result by 100to yield the percentage of sequence identity. Gaps introduced tooptimize alignment are treated as mismatched, whether introduced in thereference sequence or the comparison sequence. Optimal alignment ofsequences for comparison maybe conducted by computerized implementationof known algorithms (e.g. GAP, BESTFIT, FASTA, and TFASTA in theWisconsin Genetics Software Package, Genetics Computer Group (GCG), 575Science Dr., Madison, Wis., or BlastN and BlastX available from theNational Center for Biotechnology Information), or by inspection.Sequences are typically compared using either BlastN or BlastX withdefault parameters.

[0037] Substantial identity of polynucleotide sequences means that apolynucleotide comprises a sequence that has at least 75% sequenceidentity, preferably at least 80%, more preferably at least 90% and mostpreferably at least 95%. Typically, two polypeptides are considered tobe substantially identical if at least 40%, preferably at least 60%,more preferably at least 90%, and most preferably at least 95% areidentical or conservative substitutions. Sequences are preferablycompared to a reference sequence using GAP using default parameters.

[0038] Polypeptides that are “substantially similar” share sequences asnoted above except that residue positions which are not identical maydiffer by conservative amino acid changes. Conservative amino acidsubstitutions refer to the interchangeability of residues having similarside chains. For example, a group of amino acids having aliphatic sidechains of amino acids having aliphatic-hydroxyl side chains is serineand threonine; a group of amino acids having amide-containing sidechains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups include but are not limited to:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, asparagine-glutamine, and aspartate-glutamate.

[0039] Another indication that polynucleotide sequences aresubstantially identical is if two molecules selectively hybridize toeach other under stringent conditions. Stringent conditions are sequencedependent and will be different in different circumstances. Generally,stringent conditions are selected to be about 5° C. lower than thethermal melting point (Tm) for the specific sequence at a defined ionicstrength and pH. The Tm is the temperature (under defined ionic strengthand pH) at which 50% of the target sequence hybridizes to a perfectlymatched probe. Typically stringent conditions for a Southern blotprotocol involve washing at 65° C. with 0.2×SSC.

[0040] The present inventors have disclosed the full-length cDNAencoding human protein phosphatase methylesterase-1, termed PME-1herein.

[0041] Two PP2A C subunit mutants with single amino acid changes intheir active site residues were found to form stable complexes withcellular proteins. Mutation of either of two histidines predicted to bein the PP2A C subunit active site results in a stable complex betweenthe mutant C subunit and a protein of 44 kDa. This 44 kDa protein(formerly called p44A) is termed PME-1 herein. Immunoaffinitypurification of C subunit/PME-1 complexes generated sufficient proteinfor microsequencing of HPLC purified PME-1 tryptic peptides. Three ofthe nine peptide sequences matched a human Expressed Sequence Tag (EST),which the present inventors teach consists of the 3′ end of the PME-1coding region and the entire 3′ untranslated sequence. The completecoding region of the human PME-1 cDNA was obtained via an approachinvolving nested and semi-nested polymerase chain reaction (PCR),utilizing 3′ primers corresponding to PME-1 EST sequence and 5′ primerscorresponding to vector sequence flanking inserts in cDNA libraries. ThePME-1 protein was identified as the PP2A methylesterase by severalcriteria, including molecular size, presence of a motif found inesterases (including lipases) utilizing serine as the nucleophiliccatalytic residue, ability of okadaic acid (a known inhibitor of bothPP2A and the PP2A methylesterase) to inhibit association of PME-1 withthe C subunit mutants and to inhibit PME-1 activity, and finally,activity assays performed in vitro with bacterially expressed protein.Complex formation of PME-1 and mutant C subunit involves, at least inpart, the C subunit carboxy terminus. A catalytically inactive C subunitlacking the carboxy-terminal 9 amino acids showed decreased associationwith the methylesterase, and an antibody specific for the C subunitC-terminus, whose binding is sensitive to mutation of tyrosine 307,interfered with PME-1 binding. Finally, the two mutants that complexwith PME-1 do not bind substantial amounts of B subunit. However, twoother catalytically inactive mutants that do not bind PME-1 also aredeficient in B subunit binding.

[0042] The carboxy terminus of the protein phosphatase 2A (PP2A)catalytic (C) subunit is highly conserved. Seven of the last nineresidues (301-309) are completely invariant in all known PP2As. Includedin these invariant residues are the known pp60^(c-src) phosphorylationsite, tyrosine 307, and the known site of methylation, leucine 309.Additionally, one or more of the nine carboxy terminal residues isnecessary for formation of PP2A heterotrimers containing the Bregulatory subunit. The importance of this tyrosine for binding themethylesterase, the same change in which did not dissociate B subunit,suggests that this is the reason it is so highly conserved.

[0043] In order to create catalytically inactive PP2A C subunit mutantsthat retained maximum structural integrity, single residues likely to beinvolved in catalysis were mutated conservatively. To identify residuespotentially involved in catalysis, an alignment of PP2A and variousrelated phosphatases was performed to identify highly conservedresidues. A small number of residues were found that are identical inPP2A, PP1, PPX, PP2B, and PPλ. Of those, two histidines (H) at positions57 and 118 were chosen as having catalytic potential, and wereindividually mutated to glutamine (Q), yielding the mutants H57Q andH118Q. Subsequent to the construction of these mutants, the crystalstructures of PP1 and PP2B [Goldberg, J. et al. (1995) Nature376:745-753; Kissinger, C. R. et al. (1995) Nature 378:641-644] and amutational analysis of PPλ [Zhuo, S. et al. (1994) J. Biol. Chem.269:26234-26238] were reported, the results of which implicated thesetwo histidines in PP2A catalysis. As described herein below, each Csubunit mutant cDNA was constructed with the hemagglutinin (HA) tag atits amino terminus to allow for immunoprecipitation analysis [Ogris, E.et al. (1997) supra]. Individual mutants, wild-type C subunit, or norecombinant C subunit (vector only) were expressed stably in NIH3T3 celllines with and without coexpression of MT. In the MT expressing cells,most PP2A complexes still contain B subunit because MT is produced at alow level relative to PP2A.

[0044] After construction of stable lines, the C subunit mutants werecharacterized with respect to two properties: 1) ability to formcomplexes containing the A and B subunits or MT and 2) catalyticactivity. To examine complex formation in vivo, immunoprecipitates ofepitope-tagged wt and mutant C subunits were probed by immunoblottingfor the presence of additional subunits and MT (FIG. 1). Both mutantsbind substantial A subunit. H118Q also binds a small amount of Bsubunit, while H59Q binds almost none of this subunit. Although a smallamount of MT was found in control immunoprecipitates, levels of MT wellabove this were readily detected in the mutant immunoprecipitates,indicating that A/C/MT trimeric complexes had been formed by theseproteins. A portion of the MT coimmunoprecipitated with H59Q is shiftedrelative to the MT associated with wt C subunit; this result isreproducible and will be described in more detail elsewhere. Theseresults indicate that both of these mutants have substantial nativestructure in vivo.

[0045] To test for catalytic activity, phosphatase assays were performedon anti-tag immunoprecipitates from the various cell lines. Using bothphosphorylase and histone H1 as substrates, only wt C subunitimmunoprecipitates were found to have increased activity as compared tocontrol immunoprecipitates prepared from a cell line containing only“empty” vector (Table 1). Immunoprecipitates of the two mutants showedno activity over background towards either substrate. This finding isconsistent with previous published results for mutation of thecorresponding residues in related phosphatases.

[0046] Catalytically inactive mutants have the potential to form stablecomplexes with physiological substrates. To determine if novel cellularproteins associated with one or both catalytically inactive C subunitmutants, anti-tag immunoprecipitates were prepared from ³⁵S-labeledcells. FIG. 2A shows that, in addition to the presence of the C and Asubunits, a protein of 44 kDa (p44B) is present in theimmunoprecipitates of both catalytically inactive mutants. More p44Bappears to associate with H59Q than with H118Q. This protein is notpresent in immunoprecipitates prepared from either cells expressing wt Csubunit or cells containing only “empty” vector. The p44B proteinmigrates slightly slower than the non-specific actin band which can beseen in all lanes, and actually overlaps the actin bands in this gel. Ontwo-dimensional (2D) gels, however, p44B is completely separated fromactin and forms a streak with a pI near 7.

[0047] In order to see if sufficient p44B could be obtained tofacilitate microsequencing, scaled up immunoprecipitates were analyzedon 2D gels and silver-stained. FIG. 2C shows silver-stained 2D gels ofimmunoprecipitates from vector only control cells (GRE only) and fromcells expressing H118Q. P44B was not readily visible in these gels (seebrackets); however, another 44 kDa protein was seen that alsospecifically coimmunoprecipitates with H118Q. This protein, nowdesignated PME-1, was present in almost a 1:1 stoichiometry with the Aand C subunits and was formerly called p44a because its pI,approximately 6, was more acidic than that of p44B. A similar PME-1 spotwas found in silver-stained immunoprecipitates of H59Q. Comparison ofthe H118Q panels in FIG. 2C and FIG. 2B fails to reveal an ³⁵S-labeledspot corresponding to PME-1, suggesting that PME-1 probably has a muchlonger half-life than the PP2A C or A subunits or p44B.

[0048] To facilitate cloning of the nucleotide sequence encoding PME-1,sufficient PME-1 protein for microsequencing was obtained by purifyingepitope-tagged H59Q complexes on an anti-tag immunoaffinity column asdescribed hereinbelow. Because PME-1 migrated close to actin on standard10% SDS-PAGE, the separation of these two proteins was optimizedempirically, resulting in the use of a lower percent acrylamideelectrophoresed for an extended period of time. Proteins in the gel wereelectrophoretically transferred to PVDF membrane and visualized bystaining with Ponceau S. Both the actin and a clearly separated 44 kDaband migrating just above it were excised for further processing.Microsequencing of a tryptic peptide from the lower band confirmed thatit was indeed actin. Nine microsequences obtained from the 44 kDa bandmatched no known protein in GenBank, indicating that it was a novelprotein. However, a human EST sequence (H12112) was found deposited thatmatched three of the partial sequences obtained from the 44 kDa protein.In addition, homologous sequences were found in Caenorrhabitis eleganscosmids, and a single Saccharomyces cerevisiae homolog was identified.Additional DNA sequencing of this EST revealed coding information fortwo more PME-1 microsequences, and it was determined that H112112encoded most of the carboxy-terminal half of the PME-1 protein (162amino acids). Because the EST came from an oligo dT-primed cDNA library,it likely contains the entire 3′ untranslated region (UTR).

[0049] To obtain the missing 5′ portion of the coding region, nested andseminested PCR was performed as described in the Examples hereinbelow.5′ primers corresponded to vector sequence that flanked cDNA inserts inthe library being used as template, and 3′ primers corresponded to knownsequence (EST or newly derived 5′ sequence). In this manner, theremainder of the coding region and a portion of the 5′ UTR wereobtained. Because of the possibility of PCR errors during the multiplereamplification reactions that were necessary to obtain the completecDNA, the sequences of selected portions of the cDNA sequence wereverified. For this purpose, RT-PCR was performed with 5′ and 3′ UTRprimer sequences to generate directly from HeLa cell mRNA a productcontaining the entire coding region and much of the 5′ UTR. The finalcDNA sequence is shown in Table 2.

[0050] A schematic of a PME-1 cDNA that includes the end of the 3′ UTRdeduced from overlapping ESTs is shown in FIG. 3A. The complete cDNA isapproximately 2500 nucleotides in length, including an 1164 nucleotideregion (including tandem stop codons) encoding a protein of 386 aminoacids and a predicted pI of 5.8. All nine tryptic peptide microsequencesobtained from the purified 44 kDa band are found encoded in the clonedcoding sequence throughout its length (underlined in Table 2),confirming that this is the cDNA for the purified 44 kDa associatedprotein. This result is also consistent with the reading frame beingcorrect throughout. There is an in frame stop codon a short distance 5′of the first ATG that was verified in the RT-PCR product, so (withoutwishing to be bound by theory), we believe there is no missing 5′ codingsequence. In addition, the entire coding sequence, including thepositions of the stop codon(s), has been verified several times. Over98% of the microsequenced murine residues (107 of 109) were identical tothe human sequence. The double underlined serine at position 42corresponds to a threonine in murine PME-1. When a probe specific formouse PME-1 was used to detect transcripts from different mouse organs,a single transcript of ˜2.6 Kb was detected in all tissues (FIG. 3B). Todate, multiple ESTs have been deposited which encode portions of PME-1.These sequences separately cover the entire 3′ and 5′ UTRs, but not theentire coding region, and there is no association between the ESTsequences and the function of the encoded protein. Information from theNCBI Cancer Genome Anatomy Project (CGAP) indicates that PME-1 ESTs havebeen mapped to human chromosome 11, interval D11S916-D11S911 (80-84cM).It is not known at this time whether PME-1 is mutated in any of thediseases with defects mapped to this general region of chromosome 11.

[0051] The 386 amino acid PME-1 protein product encoded by the humanPME-1 cDNA ORF is shown in Table 2. It has a pI of 5.8, consistent withits migration on 2D gels like the one shown in FIG. 2C. All nine mousePME-1 tryptic peptide sequences (underlined in Table 2) were accountedfor in the human sequence with differences present only at a fewpositions, indicating that PME-1 is well conserved between these twospecies. Using the NCBI BLAST program, highly homologous sequencesprobably corresponding to PME-1 homologs were found for zebrafish, forC. elegans, and for S. cerevisiae. The hypothetical 88.4 kDa C. elegansprotein in chromosome 3, B0464.7, contains some of the C. eleganssequence homologous to PME-1, but lacks other highly homologoussequences, suggesting that it may represent an inaccurate prediction ofexon combinations. A more likely combination of exons that includes allB0464 cosmid exons homologous to PME-1 generates a protein of 365 aminoacids and approximately 40 kDa (Table 2). S. cerevisiae PME-1 (Table 9,Table 3) appears to be a single hypothetical 44.9 kDa protein (PIRaccession number S46814; SwissProt accession number P38796) of unknownfunction encoded by an ORF on chromosome 8R (YHN5; GenBank accessionnumber U10556). Recently YHN5 was proposed to be a mitochondrialribosome subunit protein and named YmS2, based, on a single partiallyhomologous nonapeptide sequence [Kitakawa, M. et al. (1997) Eur. J.Biochem. 245:449-456]. Human PME-1 has approximately 40% and 26%respective amino acid identity with the C. elegans and yeast sequences(Table 9). A highly charged stretch of amino acids is present in humanPME-1 but absent in PMEs from C. elegans and S. cerevisiae. This stretchof amino acids does not represent a cloning artifact, because 2D gelcomigration experiments showed that ³⁵S-labeled PME-1 in vitrotranscription/translation product comigrated precisely on 2D gels withPME-1 from HeLa cell lysates.

[0052] In order to facilitate further experiments characterizing PME-1,an anti-PME-1 peptide antibody was raised to a sixteen amino acidpeptide sequence encoded by the PME-1 cDNA. This peptide antibodydetected a 44 kDa protein present in H59Q immunoprecipitates, but absentfrom immunoprecipitates of wild-type C subunit (FIG. 4). Thus, PME-1,like p44B, associates stably with the catalytically inactive mutant Csubunits, but not with wt C subunit. Because B subunit, but not MT,requires the C subunit carboxy-terminus for association with the PP2AA/C heterodimer, we wanted to determine if MT expression might increasethe amount of PME-1 bound to H59Q. Similar levels of PME-1 werecoimmunoprecipitated from untransformed NIH3T3 cells and polyomavirusMT-transformed NIH3T3 cells (FIG. 4), indicating that MT expression doesnot greatly affect the level of H59Q/PME-1 complex formation in thecell.

[0053] When the human, C. elegans, and S. cerevisiae PME-1 proteinsequences were analyzed for motifs found in the Prosite database usingDNASTAR Lasergene software, a consensus sequence([LIV]-x-[LIVFY]-[LIVST]-G-[HYWV]-S-x-G-[GSTAC]) (SEQ ID NO:15) forlipases utilizing an active site serine was found to be conserved. Theinvariant serine in this motif, corresponding to serine 156 in humanPME-1, is the active site serine in these enzymes. In addition,scattered similarities can be seen between other regions of the PME-1sequence and some of the lipases that have this motif. Therefore, PME-1is probably a lipase whose active site serine is serine 156.

[0054] The various lipases that share this motif are found in bothprokaryotes and eukaryotes and include, among others, two D.melanogaster carboxylesterases. In addition, CheB, a bacterial glutamatemethylesterase, has a similar, but not identical, sequence surroundingits active site serine [Krueger, J. K. et al. (1992) Biochim. Biophys.Acta. 1119:332-326] (Table 8). CheB [West, A. H. et al. (1995) J. Mol.Biol. 250:276-290] and other lipases utilizing an active site serine[e.g. Winkler, F. K. et al. (1990) Nature 343:771-774; Brady, L., et al.(1990) Nature 343:767-770] have a catalytic triad in their primarysequence in the order Ser-Asp(or Glu)-His. Of the conserved histidinesin human PME-1, His 349 is a likely candidate for a putative catalytictriad histidine (Table 9). Identification of a putative PME-1 catalytictriad acidic residue by sequence comparison is more problematic becausethere are multiple acidic residues conserved between species. However,of these, two aspartates in human PME-1, Asp 181 and Asp 324, showconservation in position with putative catalytic triad aspartates inother lipases, and therefore may be more likely possibilities.

[0055] A PP2A C subunit carboxyl methylesterase of 46 kDa has recentlybeen purified [Lee, J. et al., (1996) Proc. Natl. Acad. Sci., USA93:6043-6047] but no sequence information was reported. To test thepossibility that PME-1 might be a PP2A methylesterase, PME-1 wasexpressed in bacteria and bacterial lysates were tested formethylesterase activity towards PP2A C subunit as described in theExamples herein. The results shown in FIG. 5 demonstrate that lysates ofbacteria expressing PME-1 contain a PP2A methylesterase activity notfound in bacterial lysates lacking PME-1. Similar results were obtainedwith purified recombinant PME-1 (FIG. 5). These results indicate thatPME-1 is indeed a PP2A methylesterase. Because its specificity towardsother methylated phosphatases (such as PPX) has not been characterized,it was generically named Protein Phosphatase Methylesterase-1 (PME-1).

[0056] The 46 kDa PP2A methylesterase reported by Lee and coworkers wasinhibited by okadaic acid, a potent PP2A inhibitor, but not by PMSF, acovalent inhibitor of certain serine esterases. To determine if PME-1displays similar sensitivities to these inhibitors, the abovedemethylation assay was also conducted in the presence of okadaic acidand PMSF (FIG. 5). The methylesterase activity of bacterially expressedPME-1 was inhibited by 0.1 or 1 μM okadaic acid but not by 1 or 5 mMPMSF, similar to the methylesterase purified by Lee et al. (1996) supra.

[0057] Because single amino acid changes in the C subunit active sitewere capable of inducing stable complex formation of C subunit withPME-1, it was of interest to determine if PP2A inhibitors couldantagonize the H59Q/PME-1 complex. To assay for this possibility, NIH3T3cells expressing epitope-tagged H59Q C subunit were lysed in thepresence of various phosphatase inhibitors and H59Q wasimmunoprecipitated via its epitope tag. The amount of endogenous,untagged PME-1 coimmunoprecipitating in each case was assayed byblotting with anti-PME-1 antibody (FIG. 6A). hihibitors to which PP2A ishighly sensitive (okadaic acid, sodium fluoride, and sodiumpyrophosphate), but not those to which PP2A is less sensitive orinsensitive (vanadate and phenylarsineoxide, respectively), decreasedthe amount of PME-1 bound to H59Q.

[0058] A PP2A methylesterase might be expected to make importantcontacts with carboxy-terminal residues. However, Lee and coworkersfound that PP2A carboxy-terminal peptides functioned neither asinhibitors nor as substrates for their 46 kDa PP2A methylesterase,suggesting that, at a minimum, contacts with other parts of the Csubunit are essential. To investigate the importance of the H59Q Csubunit carboxy-terminus for stable interaction with PME-1, a doublemutant, H59Q/301Stop, was created. This mutant combines the H59Qmutation, which induces stable binding of PME-1, with a deletion of thenine C subunit carboxy-terminal acids, 301-309. FIG. 6B shows theresults of an immunoprecipitation assay measuring the relative abilitiesof H59Q and H59Q/301Stop to bind A subunit and PME-1. Deletion ofresidues 301-309 from wt C subunit has previously been found to decreasethe amount of A subunit bound [Ogris, E. et al. (1997) supra]. FIG. 6Bshows that deletion of these residues from H59Q also reduces the bindingof the PP2A A subunit to H59Q. In addition, although similar amounts ofH59Q and H59Q/301Stop were immunoprecipitated in this experiment, thedouble mutant bound less PME-1 than did H59Q, indicating that one ormore of the deleted carboxy-terminal residues is important forH59Q/PME-1 complex formation. PME-1 binding was not completelyabolished, however, demonstrating that interactions also exist betweenPME-1 and other residues in the C subunit.

[0059] To address the same question via a different approach, we assayedvia immunoprecipitation whether antibodies directed against the Csubunit carboxy-terminus would compete with PME-1 for binding to H59Q.If an antibody competes with PME-1 for binding to residues on H59Q thatare important for PME-1 association, that antibody would be expected tocoimmunoprecipitate reduced amounts of PME-1 with H59Q when compared toan antibody that does not compete with PME-1. The carboxy-terminal Csubunit monoclonal antibodies used for this experiment, 1D6, 4B7, and4E1, were recently generated against a 15-residue unmethylatedcarboxy-terminal peptide. These antibodies are unable to efficientlyrecognize a C subunit mutant lacking the carboxy-terminal leucine,indicating that they bind, at least in part, at the verycarboxy-terminus. A positive control monoclonal antibody, 12CA5,immunoprecipitates H59Q via its amino-terminal epitope tag and shouldnot interfere with interactions at the C subunit carboxy-terminus[Ogris, E. et al. (1997) supra]. Comparison of the relative ratios ofthe PME-1 and H59Q bands in FIG. 6C reveals that, relative to 12CA5, 1D6and 4B7 immunoprecipitate less PME-1 for the same amount of H59Q Csubunit (the band of endogenous, wt C subunit immunoprecipitated by thecarboxy-terminal antibodies can be ignored as wt C subunit does notassociate stably with PME-1). Furthermore, although 4E1immunoprecipitated a substantial amount of H59Q C subunit (withinapproximately two-fold of 12CA5), no PME-1 could be detected, even onlong exposures. These results thus further substantiate the conclusionsmade from FIG. 6B. In addition, the fact that 1D6 and 4B7coimmunoprecipitate similar amounts of PME-1, but dramatically differentamounts of A subunit indicates that PME-1 binding does not appear to bedependent on A subunit binding.

[0060] The successful identification of the first of a number ofcellular proteins that stably associate with catalytically inactive PP2AC subunit mutants, but not with wt C subunit, is reported herein. Twoproteins of 44 kDa that differ in their isoelectric points, PME-1 andp44B, uniquely associated with two different catalytically inactive Csubunit mutants substituted individually at two different active sitehistidine residues. PME-1 was affinity purified and a cDNA encoding itwas cloned. This protein was identified as a PP2A methylesterase byseveral criteria including 1) molecular size; 2) the presence of a motiffound in lipases that use serine as their nucleophilic catalyticresidue; 3) activity assays performed in vitro with bacteriallyexpressed protein; and 4) the ability of okadaic acid, a known inhibitorof both PP2A and the PP2A methylesterase, to inhibit its activity anddecrease its association with the catalytically inactive C subunitmutant, H59Q.

[0061] Based on its molecular size, sensitivity to okadaic acid, and thelack of effect of PMSF on PME-1 activity, PME-1 is likely to beequivalent to the 46 kDa PP2A methylesterase whose purification andinitial characterization was recently reported by Lee and colleagues[Lee, J. et al.(1996) supra]. Its insensitivity to PMSF indicates thatit is not the PMSF-sensitive serine esterase/protease activity reportedby Xie and Clarke [Xie, H. et al. (1994) Biochem. Biophys. Res. Commun.203:1710-1715] which also could remove PP2A carboxymethyl groups. Leeand coworkers (1993 supra) reported that their purified PP2Amethylesterase eluted as two different peaks from an anion exchangecolumn, consistent with either differential modification or theexistence of two closely related isoforms of the enzyme. The amounts ofthese two species were within several fold of each other. Two pieces ofevidence from our studies support the idea that those two forms probablyrepresent differentially modified forms of the enzyme. First, probing ofthe GenBank EST database with the PME-1 cDNA sequence provides noevidence for a closely related PME-1 isoform, even though numerous ESTsare found which correspond precisely to the PME-1 cDNA sequence. Second,Northern blot analysis yielded a single band in multiple organs. Inaddition, we have found via immunoblotting that mammalian PME-1 in celllysates migrates on two-dimensional gels as two spots differing in theirisoelectric point in a manner consistent with a single chargedifference.

[0062] The molecular basis of the cell cycle-dependent regulation ofPP2A C subunit methylation is unknown. The poor metabolic labeling ofPME-1 in an asynchronous population of cells relative to a number ofother proteins suggests that this protein is quite stable. This resultargues against the possibility that cell cycle PP2A methylation isregulated by modulating the amount of the PP2A methylesterase. WhetherPME-1 activity is regulated is unknown. In the case of the bacterialchemotactic response, the CheB methylesterase is regulated byphosphorylation [Wylie, D. et al. (1998) Biochem. Biophys. Res. Commun.151:891-896; Hess, J. F. et al. (1998) Cell 53:79-87], while themethyltransferase is thought to be constitutively active. Lee andcoworkers (1996 supra) found no difference in the activity of their twopurified forms of PP2A methylesterase, suggesting that the differentialmodification likely responsible for generating these two forms might notbe involved in regulation of activity of this enzyme. It is possible,however, that effects might be seen under other conditions, or that anadditional protein(s) may be necessary for an effect to be manifested.In addition, it is possible that more than one modification occurs.

[0063] Without wishing to be bound by any particular theory, it isbelieved that PP2A methyltransferase and methylesterase enzymes achievetheir specificity, in part, by interacting with or near the active siteof the PP2A C subunit. It was reported previously that neither the PP2Amethyltransferase nor the PP2A methylesterase can recognize shortpeptide substrates corresponding to the C subunit carboxy-terminus.Thus, functional recognition by both these enzymes requires additional Csubunit structure. Additionally, as demonstrated in this study,perturbation of the C subunit active site by either of two differentmutations can stabilize the interaction with the PME-1 methylesterase.Furthermore, PP2A inhibitors have a destabilizing effect on thePME-1/H59Q interaction. Finally, the methyltransferase is inhibited bythe PP2A inhibitors, okadaic acid and microcystin-LR, and themethylesterase is inhibited by okadaic acid (testing for inhibition ofthe methylesterase by microcystin has not been reported). Although ithas been proposed that this inhibition is due to the interaction ofthese inhibitors with carboxy-terminal C subunit residues, the PP2Ainhibitors, sodium fluoride or sodium pyrophosphate, partially or fullydisrupt PME-1 /H59Q complexes. The latter effect is more consistent witha role in binding the PME-1 methylesterase for active site residuesand/or metals, or nearby residues sensitive to effects on the activesite. Four separate catalytically inactive PP2A active site pointmutants including the two described in this study, are methylated atless than 3% of the wild-type level in vivo and in vitro. Although webelieve there is interaction with residues and/or metals in or near theactive site, but another equally viable possibility is that mutation ofactive site residues and/or binding of inhibitors has more distanteffects on the C subunit conformation critical for stable complexformation with PME-1.

[0064] Contact between the C subunit and PME-1 could be with PME-1residues and/or with a phosphorylation site on PME-1. Because H59Q andH118Q are virtually unmethylated, PME-1 apparently can remain bound tothese mutants in the absence of a methylated carboxy-terminus. At leastwith H59Q, PME-1's contacts other than on the C subunit carboxy-terminusare strong enough to result in substantial complex formation in theabsence of the nine carboxy-terminal C subunit residues. This conclusionis further supported by the finding that two C subunit carboxy-terminalpeptide antibodies, known to require Leu 309 for efficient binding,immunoprecipitate H59Q/PME-1 complexes. However, the amount of PME-1coimmunoprecipitated by these antibodies was less than thatcoimmunoprecipitated by an antibody recognizing an amino-terminalepitope tag on the C subunit. The latter result and the fact that athird carboxy-terminal C subunit antibody could not immunoprecipitateH59Q/PME-1 complexes at all suggest that PME-1 is proximal to the Csubunit carboxy-terminus in the H59Q/PME-1 complex. Moreover, thereduced amounts of PME-1 in complex with the H59Q/301Stop double mutantindicate that carboxy-terminal residues play a role in binding of H59Qto PME-1. The contribution of these residues to the interaction of wildtype C subunit with PME-1 might be even more important in the absence ofthe complex-stabilizing H59Q mutation.

[0065] The decreased B subunit binding observed with these mutants mightbe due indirectly to lack of methylation at the carboxy-terminus ofthese mutants. The fact that H59Q and H118Q bind the structural PP2A Asubunit and polyomavirus MT suggests that they are not grossly alteredin their structure. Two other catalytically inactive point mutants thatbind A subunit and polyomavirus MT, but are highly deficient inmethylation are also deficient in B subunit binding. Given that the Bsubunit requires the C subunit carboxy-terminus for stable complexformation with the A/C heterodimer, the B subunit might require amethylated carboxy-terminus for efficient binding to C subunit. Analternate, but not mutually exclusive, possibility is that thecarboxy-terminus and the active site are proximal in the threedimensional structure of the C subunit. This model would provide anexplanation for how events occurring at the carboxy-terminus (B subunitbinding, methylation, phosphorylation, etc.) can affect the active site(activity, specificity), and vice versa. In addition, at least for H59Qand H 118Q, PME-1 and B subunit binding might be mutually exclusive.

[0066] These catalytically inactive C subunit mutants should be usefulfor identifying other proteins involved in PP2A signaling. H59Q andH118Q bind multiple proteins not bound stably by wt C subunit. Theseinclude, in addition to PME-1, p44B and other proteins not marked, butvisible, in FIG. 2B. Interestingly, initial experiments suggest thatp44B binding to H59Q is even more sensitive to phosphatase inhibitorsthan is PME-1 binding. These proteins could be PP2A substrates or otherproteins whose binding is sensitive to the state of the C subunit activesite. One of these proteins is the same molecular size as the PP2Amethyltransferase reported by Lee and colleagues (Lee et al. (1993)supra]. Catalytically inactive mutants of dual specificity and tyrosinephosphatases [Gelerloos, J. A., et al. (1996) Oncogene 13:2367-2368;Bliska, J. B. et al. (1992) J. Exp. Med. 176:1625-1630] have beenpreviously used successfully to identify novel substrates, but unlikePP2A, their catalytic mechanisms involve the formation of covalentintermediates with substrates.

[0067] PME-1 and p44B differ in several characteristics, suggesting thatthese two proteins are not simply modified forms of one another. Theyare separated from each other on two-dimensional gels by approximatelyone pH unit, which is unlikely to be accounted for by modification;PME-1 forms sharp spots on these gels while p44B migrates as a smear. Inaddition, in vitro translation of PME-1 yields no product migrating atthe position of p44B and we have been unable to detect p44B withantibodies raised against PME-1 sequences.

[0068] Finally, because of the high conservation of PP2A with otherphosphatases such as PP1, PPX, PPV, etc., it will be of interest to seeif similar or different cellular proteins bind stably to thesephosphatases when the residues corresponding to PP2A H59 and H118 aremutated to glutamine. One question of special interest is whether thecorresponding catalytically inactive mutants of PPX, which has the samelast four carboxy-terminal amino acids as PP2A and is also methylated atits carboxy-terminal leucine, will also trap PME-1.

[0069] The present invention provides the coding sequences for themammalian PME1 protein, as specifically exemplified by the human codingsequence. This allows the construction of recombinant DNA molecules andrecombinant host cells produced in the laboratory, which molecules andhost cells are used for the recombinant expression of the PME1 proteinand enables assay methods for determining inhibitors of themethylesterase activity of the PME-1 protein, and thus, compounds whichslow the growth of cells, especially neoplastic and/or transformedcells.

[0070] Without wishing to be bound by theory, the present inventorspropose that the protein of the present invention is a ProteinPhosphatase Methylesterase-1 (PME-1) which removes methyl groups fromthe PP2A growth-regulating protein phosphatase, and that the methylationstatus of the catalytic subunit affects activity and thus plays a rolein growth regulation and normal progression of the cell cycle. See,e.g., Lee et al. (1996) supra, for a description of the methylesteraseand methods for assay.

[0071] Monoclonal or polyclonal antibodies, preferably monoclonal,specifically reacting with the methylesterase of the present inventionencoded by a particular coding sequence may be made by methods known inthe art. See, e.g., Harlow and Lane (1988) Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratories; Goding (1986) MonoclonalAntibodies: Principles and Practice, 2d ed., Academic Press, New York.

[0072] Standard techniques for cloning, DNA isolation, amplification andpurification, for enzymatic reactions involving DNA ligase, DNApolymerase, restriction endonucleases and the like, and variousseparation techniques are those known and commonly employed by thoseskilled in the art. A number of standard techniques are described inSambrook et al. (1989) supra; Maniatis et al. (1982) Molecular Cloning,Cold Spring Harbor Laboratory, Plainview, N.Y.; Wu (ed.) (1993) Meth.Enzymol. 218: Part I; Wu (ed.) (1979) Meth Enzymol. 68; Wu et al. (eds.)(1983) Meth. Enzymol. 100 and 101; Grossman and Moldave (eds.) Meth.Enzymol. 65; Miller (ed.) (1972) Experiments in Molecular Genetics, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.; Old and Primrose(1981) Principles of Gene Manipulation, University of California Press,Berkeley; Schleif and Wensink (1982) Practical Methods in MolecularBiology; Glover (ed.) (1985) DNA Cloning Vol. I and II, IRL Press,Oxford, UK; Hames and Higgins (eds.) (1985) Nucleic Acid Hybridization,IRL Press, Oxford, UK; and Setlow and Hollaender (1979) GeneticEngineering: Principles and Methods, Vols. 1-4, Plenum Press, New York.Abbreviations and nomenclature, where employed, are deemed standard inthe field and commonly used in professional journals such as those citedherein.

[0073] All references cited in the present application are incorporatedby reference herein to the extent that they are not inconsistent withthe present Specification.

[0074] The following examples are provided for illustrative purposes,and are not intended to limit the scope of the invention as claimedherein. Any variations in the exemplified sequences and methods whichoccur to the skilled artisan are intended to fall within the scope ofthe present invention.

EXAMPLES Example 1

[0075] Plasmids and Mutagenesis

[0076] Site-directed mutagenesis was performed on a HA-tagged wt Csubunit cDNA cloned in the pcDNA I Amp vector [Ogris et al. (1997)supra] using the Muta-Gene Phagemid In Vitro Mutagenesis Kit accordingto the manufacturer's instructions (Bio-Rad Laboratories, Hercules,Calif.). The entire cDNA of both H59Q and H118Q was sequenced to confirmsuccessful mutagenesis and to ensure that no additional mutationoccurred. Mutant C subunit cDNAs including the HA tag coding sequencewere cloned into the dexamethasone-inducible vector, pGRE 5-2 [Mader,S., and White, J. H. (1993) Proc. Natl. Acad. Sci. USA 90:5603-5607].The construction of a pGRE5-2 vector expressing HA-tagged wt PP2A Csubunit has been previously described [Ogris et al. (1997) supra]. Aninducible vector was chosen to try to minimize the potential deleteriouseffects of wild-type and mutant C subunits (if any) while lines werebeing carried in culture, and to provide for an uninduced control inanalyses of their effects.

Example 2

[0077] Cells and Cell Culture

[0078] NIH 3T3 lines expressing wt polyomavirus MT and a geneticinresistance gene [Cherington et al. (1986) Proc. Nat. Acad. Sci. USA83:4307-4311] were transfected by the calcium phosphate precipitationmethod [Sambrook et al. (1989) supra], and individual clones andmixtures of clones expressing wt C subunit (36wt), H59Q, H118Q, or emptyvector (GREonly) were selected and maintained as described previously[Ogris et al. (1997) supra]. H118Q expressed at a level well below thatof endogenous wt C subunit, while H59Q expressed at a level equal to orgreater than the wt level. Although the inducible vector, pGRE5-2, wasused to express these proteins, their levels were substantial in theabsence of dexamethasone; for this reason, GREonly cells were used as anegative control in this study rather than uninduced wt or mutant Csubunit expressing cells. However, dexamethasone treatment was alwaysused to obtain maximal expression of the C subunits.

Example 3

[0079] Radiolabeling of Cells

[0080] For metabolic labeling of cells with methionine, subconfluentdishes of cells were labeled for 5h with [³⁵S] methionine (300 uCi/ml)in DMEM minus methionine supplemented with 0.5% dialyzed fetal bovineserum.

Example 4 Preparation of Cell Lysates and Immunopreciptation

[0081] The details of treating the cells with dexamethasone, preparationof cell lysates, and immunoprecipation of C subunits have been describedpreviously [Ogris et al. (1997) supra]. For experiments quantitatingPME-1 binding to different mutants (FIG. 6B), immunoprecipates werewashed twice with NP40 lysis buffer, twice with PBS, and once withddH20. Washed immune complexes were used for phosphatase assays oranalyzed by one or two-dimensional gel electrophoresis.

Example 5

[0082] One- and Two-Dimensional Gel Electrophoresis and Fluorography

[0083] SDS-polyacrylamide gel (10% acrylamide) was performed accordingto Laemmli [Laemmli, U. K. (1970) Nature 227:680-685]. Gels were silverstained by the procedure of Wray et al. [Wray, W. et al. (1981)Biochemistry 118:197-203] except that after electrophoresis the gelswere sequentially incubated 10 min in distilled water (200 ml), 10 minin 95% ethanol (200 ml), 1 h. in 50% methanol (100 ml), and 30 min indistilled water (100 ml) prior to staining.

Example 6

[0084] Immunoblotting

[0085] Immunoblotting [Towbin, H. et al. (1979) Proc. Natl. Acad. Sci.USA 76:4350-4354] was performed with mouse monoclonal anti-tagantibody(16B12; 1:5000 dilution of ascites; BAbCO, Richmond, Calif.);rabbit anti-B subunit antibody (#16; 1:5000); affinity-purified rabbit(R39; 1:5000) or mouse monoclonal (4G7; 1 μg/ml) anti-A subunitantibodies; mouse monoclonal anti-C subunit antibody (1D6; 0.25 μg/ml);or rabbit ant-PME-1 antibodies (AR2 or E37; see below). Immunoblots weredeveloped with enhanced chemiluminescences (Amersham, Arlington Heights,Ill.).

Example 7

[0086] Phosphatase Assay

[0087] Phosphatase activity present in anti-HA tag immunoprecipitatesfrom the different cell lines was assayed using phosphorylase a andHistone H1. [γ-³²P]-labeled phosphorylase a substrate was prepared fromphosphorylase b according to the manufacturer's (GibcoBRL, Gaithersburg,Md.) instructions. Histone H1 was phosphorylated by mitotic p34^(cdc2)purified from Nocodazole arrested HeLa cells as described [Mayer-Jaekelet al. (1994) supra]. Lysates used for immunoprecipitation wereequilibrated according to epitope-tagged C subunit expression levels.Assays were performed at a linear range and with subsaturating amountsof each substrate.

Example 8

[0088] Purification and Microsequencing of PME-1

[0089] To obtain PME-1 protein for microsequencing, H59Q C subunitcomplexes containing PME-1 were immunoaffinity purified. In total, 135confluent 15 cm dishes of MT-transformed NIH3T3 cells expressingHA-tagged H59Q were needed to obtain enough PME-1 for microsequencing.Forty-five separate immunoaffinity purifications were performed on 3dishes of lysate at a time, reusing the same immunoaffinity matrix atleast 15 times. To prepare the immunoaffinity matrix, anti-HA tagantibody (12CA5; obtained from BAbCO) was chemically crosslinked toprotein A-Sepharose beads (Pharmacia, Piscataway, N.J.) by publishedmethods [Harlow, E., and Lane, D. (1988) supra]. After washing 3 dishesof cells twice with PBS and once with IP wash (10% (vol/vol) glycerol;135 mM NaCl; 20 mM Tris, pH 8.0), the cells were scraped and lysed at 4°C. with rocking for 10 min in 1.0 ml of NP40 lysis buffer (IP washcontaining 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, and 0.03units/ml aprotinin). Lysates were cleared at 13,000×g and then incubatedfor 1 h at 4° C. while rocking with 500 μl of the crosslinkedantibody/bead complexes. Complexes were washed once with NP40 lysisbuffer, three times with Tris-buffered saline, and then twice withddH₂O. Bound H59Q complexes containing PME-1 were eluted by threesequential incubations with 300 μl of 20 mM triethylamine. Eluates werequickly frozen on dry ice and stored frozen until all batches ofaffinity purification had been completed. The antibody/bead complexeswere then washed twice with 20 mM triethylamine and twice with IPlyseprior to reuse. After H59Q complexes had been purified from all 135dishes of cells, eluates containing PME-1 were concentrated to drynessby vacuum centrifugation, and the residues were suspended in PBS and gelbuffer and analyzed on three separate SDS-polyacrylamide gels [Laemmli,U. K. (1970) Nature 227:680-685]. One-dimensional gels were chosen toavoid losses associated with 2D gel analysis. Because PME-1 migratesclosely to actin, the separation of these two proteins was maximized bythe use of an 8% SDS-polyacrylamide gel run for an extended period oftime.

Example 9

[0090] Trypsin Digestion, HPLC Separation and Microsequencing

[0091] After separation of PME-1 complexes by SDS-PAGE, the proteinswere electrotransferred to polyvinylidiene difluoride (PVDF) membraneand stained with Ponceau S. Individual protein bands were excised andsubmitted to in situ digestion with trypsin [Fernandez et al. (1994)Anal. Biochem. 218:112-117; Lane et al. (1991) J. Protein Chem.10:151-160]. The resulting peptide mixture was separated by microborehigh performance liquid chromatography using a Zorbax C18 2.1 mm by 150mm reverse phase column on a Hewlett-Packard 1090 HPLC/1040 diode arraydetector. Optimum fractions from the chromatogram were chosen based ondifferential UV absorbance at 205 nm, 277 nm and 292 nm, peak symmetryand resolution. Peaks were further screened for length and homogeneityby matrix-assisted laser desorption time-of-flight mass spectrometry(MALDI-MS) on a Finnigan Lasermat 2000 (Hemel, England); and selectedfractions were submitted to automated Edman degradation on an AppliedBiosystems 494A, 477A (Foster City, Calif.) or Hewlett Packard G1005A(Palo Alto, Calif.). Details of general strategies for the selection ofpeptide fractions and their microsequencing have been previouslydescribed [Lane et al. (1991) supra].

Example 10

[0092] cDNA Cloning via PCR and RT-PCR

[0093] To obtain the missing 5′ portion of the PME-1 coding region,nested and seminested PCR were performed using human B cell, humanhippocampus, and human kidney cDNA plasmid libraries. 5′ primerscorresponded to vector sequence that flanked cDNA inserts in the librarybeing used as template, while 3′ primers corresponded to known sequence(EST or newly derived 5′ PME-1 sequence). Southern Blotting using anend-labeled 20 bp oligonucleotide corresponding to known PME-1 sequenceupstream of the 3′ PCR primer was employed to identify authentic PME-1products after each reaction. PCR products containing 5′ extensions ofthe PME-1 sequence were purified using a PCR product purification kit(Boehringer-Mannheim, Indianapolis, Ind.), cloned, and sequenced. Newprimers were designed for PCR and Southern Blotting and then the abovesteps were repeated until the sequence of the remainder of the PME-1coding region and a portion of the 5′UTR were obtained.

[0094] Total mRNA was purified from HeLa cells using Trizol Reagent(Life Technologies, Gaithersburg, Md.) according to the manufacturer'sinstructions. RT-PCR was employed to obtain a PME-1 cDNA from HeLa cellmRNA. First strand synthesis was performed with Avian MyeloblastosisVirus reverse transcriptase (Boehringer-Mannheim, Indianapolis, Ind.) bythe manufacturer's protocol using a primer from the PME-1 3′ UTR(TGTTGAGGAGGGGTGGACAG) (SEQ ID NO:1). Using pfu polymerase (Stratagene,La Jolla, Calif.), the product was used for PCR with the same 3′ primerand a primer from the PME-1 5′ UTR (TGTATGGGGACCTTCCTCCT) (SEQ ID NO:2)to generate a cDNA containing the entire PME-1 coding region and much ofthe 5′ UTR, including the in frame stop codon upstream of the putativestart ATG.

[0095] Obtaining the entire human PME-1 coding sequence requiredhundreds of PCR reactions, scores of oligonucleotide primers, manySouthern blots and numerous subclonings and sequencing reactions. Mostlibraries did not contain cDNAs with a full length PME-1 codingsequence.

Example 11

[0096] Purification of His-Tagged PME-1 from Bacteria ExpressingRecombinant His-Tagged PME-1

[0097]E. coli (PR13Q) expressing recombinant His-tagged PME-1 from anisopropylthiogalactoside (IPTG) inducible lac promoter were grown to anO.D. at 600 nm of 0.7 and then induced with IPTG for 2-3 h. The PME-1coding sequence is fused in frame in a vector such as pThioHis A, B, C,pTrcHis A, B, C or pTrcHis 2A, B, C (Invitrogen, Carlsbad, Calif.).Cells were collected by centrifugation and broken open in the presenceof protease inhibitor by sonication or use of a French Pressure cell,using a lysis buffer containing normal saline (137 mM), and 20 mMTrisHCl (pH8.0). Lysates were cleared by centrifugation at ≧13,000×g,and supernatants were incubated in batch with a nickel-agarose columnmatrix (Chelating Sepharose, Pharmacia, Piscataway, N.J.) for 1-2 h at4° C. with rocking. Alternatively, a packed nickel-agarose column wasused and the supernatant was passed over it slowly several times. Ineither case the nickel-agarose/6×His-PME-1 complexes were washed andthen His-tagged PME-1 was eluted with increasing amounts of imidizole(either with a step or continuous gradient). PME-1 protein thus isolatedwas dialyzed to remove the imidizole or analyzed on a Mono-Q column.Milligram amounts of PME-1 protein have been obtained from a liter ofculture.

Example 12

[0098] Assay for PP2A Methylesterase Activity

[0099] Epitope-tagged PP2A C subunits with ³H-methyl groups incorporatedin vitro were immunoprecipitated with anti-tag antibody and used assubstrate for PME-1. PME-1 enzyme sources assayed include: lysates ofbacteria or baculovirus-infected Sf9 insect cells expressing His-taggedPME-1 and immunoprecipitated PME-1 from baculovirus-infected Sf9 insectcells. Control lysates from bacteria or Sf9 cells not expressingrecombinant PME-1 were also incubated with tritiated substrate tomeasure non-specific background from the lysates. After 1 h incubationat 32° C., the amount of ³H-methyl groups remaining was assayed bySDS-polyacrylamide gel electrophoresis (SDS-PAGE). PME-1 was clearlyable to demethylate PP2A C subunit as measured by this assay. TheHA-tagged PME-1 expressed in the baculovirus vector has beendemonstrated to have PP2A methylesterase activity.

[0100] In a second methylesterase assay, unlabeled epitope-tagged PP2A Csubunits were immunoprecipitated with anti-tag antibody and used assubstrate for PME-1. As PME-1 enzyme source, the following were used:cell lysates of bacteria expressing HA-tagged, His-tagged, and untaggedPME-1; cell lysates of HA-tagged or untagged PME-1-expressingbaculovirus-infected Sf9 insect cells; purified bacterial HA-taggedPME-1, purified (immunoprecipitated) baculovirus-infected Sf9 HA-taggedPME-1. Control lysates from bacteria or Sf9 cells not expressingrecombinant PME-1 were also incubated with substrate to measurenon-specific background from the lysates. After 1h incubation at 32° C.,the C subunit immunoprecipitates were washed and analyzed by SDS-PAGE.The proteins in the gels were electrophoretically transferred tonitrocellulose membranes and then the membranes were probed withmonoclonal antibody (made in our laboratory) that only recognizesunmethylated PP2A. A second probing of the same membrane with amethylation-insensitive antibody shows the actual amount of PP2A Csubunit in each lane. Comparison of the blotting signals for the twodifferent antibodies allows demethylation to be evaluated (the signal ofthe methylation-inhibited antibody gets stronger as PP2A C subunit isdemethylated). PME-1 was clearly able to demethylate PP2A C subunit, asmeasured by this assay.

[0101] An in vitro methylesterase activity assay using the PME-1protein, for example, produced as a recombinant human PME-1, can be usedto screen test compounds for inhibition of PME-1. Inhibitors of PME-1 ine.g., neoplastic cells slow the growth of those cells. Inhibitors couldalso be used to slow the growth in other hyperproliferative conditions.In Alzheimer's disease, PP2A has reduced activity. Identification ofcompounds which increase PP2A activity, for example, by appropriatelymodulating PME-1 activity, allows treatment to slow the progression ofAlzheimer's disease, and thus postpone loss of mental function inaffected patients.

Example 13

[0102] Computer Analyses

[0103] The NCBI BLAST program [Altschul, S. F. et al. (1990) J. Mol.Biol. 215:403-410] was used to probe various databases for PME-1 ESTsand related proteins. The DNASTAR Lasergene software package wasutilized for alignments and identification of the PROSITE databaselipase motif found in PME-1.

Example 14

[0104] Northern Blot

[0105] Adult Balb/c mice were sacrificed and organs removed andflash-frozen in liquid nitrogen. Total RNA from the organs was isolatedusing the RNeasy kit (QIAGEN) and analyzed on formaldehyde-1% agarosegels to check for RNA integrity and to estimate the amount of the 18Sand 28S RNAs. Based on these estimates, similar amounts of RNA wereseparated on formaldehyde-1% agarose gels and transferred to GeneScreennylon membranes. After UV-crosslinking, the membranes were stained witha 0.04% methylene blue solution to visualize the RNA. Filters were thenhybridized with a ³²P-radiolabeled probe generated by random primerlabeling of a DNA fragment from the 3′ untranslated region of the mousePME-1 cDNA. the probe, 395 bp in length, is an EcoRI-NotI fragment of aPME-1 EST clone (accession number W34856). The blots were used forautoradiography with X-ray film and/or analysed on a STORMPhosphorImager (Molecular Dynamics, Sunnyvale, Calif.).

Example 15

[0106] Production of Polyclonal Antibodies Recognizing PME-1

[0107] Two different antisera recognizing PME-1 were raised in rabbits.The first AR2, was raised against a 16-residue PME-1 peptide sequence(RIELAKTEKYWDGWFR) (amino acids 288-303, SEQ ID NO:5) found encoded inthe PME-1 cDNA. The peptide was conjugated to Keyhole Limpet Hemocyanin(KLH) via an added carboxy-terminal cysteine residue using a PieceImject conjugation kit, and the conjugate was used as immunogen. Thesecond antiserum, E37, was raised against a mixture of two-nickelagarose-purified, 6×His-tagged, bacterially expressed human PME-1fragments that together represent the carboxy-terminal half of theprotein. For each immunogen, a single female New Zealand white rabbitwas immunized and boosted multiple times using Freund's adjuvant.

Example 16

[0108] PME-1 Sequences from other Organisms

[0109] Yeast PME-1 was found by homology searches of sequence databasesusing the NCBI BLAST program and the known human PME-1 sequence. Genomicyeast PME-1 sequence was examined and found to have no introns;therefore, we designed PCR primers for yeast PME-1 carried out PCR,cloned the PCR product into a bacterial vector and sequenced it to makesure no PCR errors had occurred.

[0110] Table 3 shows the amino acid sequence of the yeast methylesterasehomolog of PME-1. Table 6 provides the coding sequence for the yeastPME-1 protein.

[0111] The C. elegans PME-1 coding sequence was deduced by homology withmammalian and yeast PME-1 sequenced. However, it should be noted thatthis gene product was not predicted by the Genefinder program.

[0112] Table 4 provides the amino acid sequence of the C. elegans PME-1homolog and Table 7 gives the coding sequence. Review of the ESTsequences revealed two potential alternative splicing scenarios. Thealternate which encoded an LLSTYCR amino acid segment (SEQ ID NO:17) wasruled out based on the lack of a similar amino acid segment in the yeastPME-1 protein and poor alignment with the human protein sequence.

[0113] Table 9 illustrates the alignment of human, C. elegans and S.cerevisiae (YHN5) PME-1 protein sequences. Residues identical with humanPME-1 are as white-on-black. Residues corresponding to the Prosite motiffor lipases employing an active site serine are boxed.

[0114] The mouse PME-1 sequences were found by search for EST sequenceson Genbank with significant homology to the human PME-1 DNA sequencesdisclosed herein. Table 5 represents a portion of the mouse codingsequence generated by homology searches and computer-aided alignment ofthe mouse sequences to the human sequences and creating a consensussequence for the nucleotides of the various homologous ESTs. The first283 nucleotides of Table 5 are from a single EST (Genbank Accession No.AA555778). The next 465 nucleotides are given as X's because there wasno mouse sequence homologous to the corresponding human PME-1 cDNAsequence. It is understood that the actual length may not be exactly 465nucleotides. The following 527 nucleotides are from a single mouse EST(Accession No. AA644991.) The next seven nucleotides (1276-1282) arefrom an overlap of AA644991 with AA672810. The following 132 nucleotidesare from AA 672810 only. Then two other ESTs overlap; thus, most of theremaining nucleotides are quite certain, with the following exceptions.The nucleotides at positions 1942-1943 are somewhat ambiguous in thattwo ESTs have the identified sequence while others have TA, TN or T-.The G at position 2167 is from 2 of 3 ESTs. R at 2169 is from a G and Ain two ESTs. The sequence at 2174-2175 appears unreliable. Nucleotides2247-2270 are from a single EST (Accession No. EST AA260585) andnucleotides 2337-2409 are from a single minus-strand EST (Accession No.T25552).

[0115] Plants also have similar growth regulatoryphosphatase-kinase-methylation-demethylation systems, and there is aplant protein having significant homology to the mouse, human, yeast andnematode (C. elegans) PME-1 sequences, especially to the catalytic andGQMQGK (amino acids 333-338 of SEQ ID NO:5) regions of human PME-1. Theplant homolog(s) of PME-1 can be identified using techniques similar tothose described herein, including, but not limited to, the use ofsequence database searches in conjunction with PCR, RT-PCR and/orhybridization studies and immunological screening with antibodiesspecific for a PME-1 protein. TABLE 1 H59Q and H118Q are catalyticallyinactive^(a) C subunit-associated phosphatase activity (% wt)^(b)phosphorylase a cdc2-phosphorylated C subunit (Means ± s.d.) Histone H1(Mean ± s.d.) None (vector 9 ± 2 2 ± 1 only control) wt 100 100 H59Q 7 ±1 2 ± 1 H118Q 8 ± 3 2 ± 1

[0116] TABLE 2 Nucleotide and Deduced Amino Acid Sequences for HumanPME-1 (SEQ ID NO:4 and SEQ ID NO:5 respectively)

[0117] TABLE 3 Saccharomyces cerevisiae PME-1 Amino Acid SequenceMSDDLRRKIALSQFEPAKNVLDATFQEAYEDDENDGDALGSLPSFNGQSNRNRKY (SEQ ID NO:6)TGKTGSTTDRISSKEKSSLPTWSDFFDNKELVSLPDRDLDVNTYYTLPTSLLSNTTSIPTFTFHHGAGSSGLSFANLAKELNTKLEGRCGCFAFDARGHAETKFKKADAPICFDRDSFIKDFVSLLNYWFKSKISQEPLQKVSVILIGHSLGGSTCTFAYPKLSTELQKKILGITMLDIVEEAAIMALNKVEHFLQNTPNVFESINDAVDWHVQHALSRLRSSAEIAIPALFAPLKSGKVVRITNLKTFSPPWDTWFTDLSHSFVGLPVSKLLILAGNENLDKELIVGQMQGKYQLVVFQDSGHFIQEDSPIKTAITLIDFWKRNDSRNVVIKTNWGQHK TVQNT

[0118] From Genbank sequence sequences identified as encoding ahypothetical protein; deposited by JOHNSTON M., ANDREWS S., BRINKMAN R.,COOPER J., DING H., DOVER J., DU Z., FAVELLO A., FULTON L., GATTUNG S.,GEISEL C., KIRSTEN J., KUCABA T., HILLIER, L., JIER M., JOHNSTON L.,LANGSTON Y., ATREILLE P., LOUIS E. J., MACRI C., MARDIS E., MENEZES S.,MOUSER L., NHAN M., RIFKIN L., RILES L., ST. PETER H., TREVASKIS E.,VAUGHAN K., VIGNATI D., WILCOX L., WOHLDMAN P., WATERSTON R., WILSON R.,VAUDIN M.; See also SCIENCE 265:2077-2082(1994).

[0119] ACCESSION: P38796; PIR: S46814 #type complete ACCESSION: S46814GB: YSCH9205 ACCESSION: U10556 DESC HYPOTHETICAL 44.9 KD PROTEIN INERG7-NMD2 INTERGENIC REGION. DATE Feb. 1, 1995 (REL. 31, CREATED) Feb.1, 1995 (REL. 31, LAST SEQUENCE UPDATE) Feb. 1, 1995 (REL. 31, LASTANNOTATION UPDATE) GENE YHR 075C. #map_position 8R COM SEQUENCE FROMN.A. STRAIN=S288C/AB972; MED MEDLIN; 94378003. AUTH TAXONOMY EUKARYOTA;FUNGI; ASCOMYCOTINA; HEMIASCOMYCETES. COMMENT Nucleic Acid Featurestranslated to generate this entry: CDS complement(9569 . . .10771)/codon_start=1/evidence=not_experimental/db_xref=“PID:g500835”TABLE 4 Caenorhabditis elegans PME-1 Amino Acid Sequence. (SEQ ID NO:7)MSDDKLDTLPDLQSETSHVTTPHRQNDLLRQAVTHGRPPPVPSTSTSGKKREMSELPWSDFFDEKKDANIDGDVFNVYIKGNEGPIFYLLHGGGYSGLTWACFAKELATLISCRVVAPDLRGHGDTKCSDEHDLSKETQIKDIGAIFKNIFGEDDSPVCIVGHSMGGALAIHTLNAKMISSKVAALIVIDVVEGSAMEALGGMVHFLHSRPSSFPSIEKAIHWCLSSGTARNPTAARVSMPSQIREVSEHEYTWRIDLTTTEQYWKGWFEGLSKEFLGCSVPKMLVLAGVDRLDRDLTIGQMQGKFQTCVLPKVGHCVQEDSPQNLADEVGRFACRHRIAQPKFSALASP PDPAILEYRKRHHQ

[0120] TABLE 5 Partial CDNA Sequence of Mus musculus PME-1 homolog (SEQID NO:8) TTGTACTGCACGTATCGTGGGACGGACCTTGGGCCACTGTTGTCGACGTGCGGCCTCCCTTTGATGTCGGCCCTTGAAAAAAGCATGCACCTCGGCCGCCTACCTTCTCGCCCTCCTCTACCCGGCAGCGGGGGCAGTCAGAGCGGACGCAAGATGCGGATGGGCCCTGGACGGAAGCGGGACTTTACCCCTGTCCCATGGAGTCAGTACTTTGAGTCAATGGAAGATGTGGAAGTGGAGAATGAAACTGGCAAGGATACTTTTCGAGTTTACAAGATTGGTTXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXTTCGGATCCTTGGCCAAGTCAAACAGTGTGAAGGAATTACAAGTCCAGAAGGTTCCAAATCCATAGTGGAAGGAATCATAGAGGAGGAGGAAGAAGATGAGGAAGGAAGTGAGTCAGTTAACAAGAGGAAAAAGGAAGACGACATGGAAACCAAGAAGGATCACCCATACACCTGGAGAATTGAGCTGGCAAAAACAGAAAAGTACTGGGATGGCTGGTTCCGGGGCTTATCCAATCTCTTTCTTAGCTGTCCTATTCCTAAACTGCTGCTCTTGGCGGGTGTTGACAGATTGGATAAAGATCTGACCATAGGCCAGATGCAGGGGAAGTTCCAGATGCAGGTCTTACCCCAGTGTGGCCATGCAGTCCATGAGGATGCCCCTGACAAGGTAGCTGAAGCTGTTGCCACTTTCCTGATCCGGCACAGGTTTGCAGAGCCCATCGGAGGATTCCAGTGTGTGTTTACTGGCTGCTAGTGACCTGCTGTCTACTCCTCCCTCTACATTGAGCTCTGTTGTAAATACATCGCACCAGAGGCCACTGTGACGCCGCTGTCTCCTCCTCTCCATCCCGCCCAGCCATGTGACACCGGCTCTTGTAGAGGGCATCCCCAGATGTCCAAACCCTTTCCTGTGTACTGTTGAAAGCATTGTTCTTCAGGGCCCTTGTCCAACAGTGGCCCGTGCAGTCTGGGGTCCACAGCTCTTCCTCTCCTTCCTGTGCTCCCTGCTTGCCTAGGATGAAGCCTCCAGCGCTGCTCCCTGGCCCTGTTCCTGGCATATGGCAATGTACCCCAGGCTCAGGGATCTCCCTTCCTTGAGGATGTTCTTGGCATGGTCCTGCCCTACCTCATGGGATGGGCAATGCACACACTGGCCCTTATTTTTCCCTTTCAAATAAAACACCAGTCAGGTACCTTTATCCCAGTCTTAACTGTCCCAAATCTGGAAGGTCCAGAGTAAGCAGGATTCAGGGAGAGGGAGTGGATAGCAAGTATCCCAAGAAACCAACCTGTAAGTCAGGTCCAGCCAGTCCAAGCACATGGCTTCCCATCTGGGTGAGCCCACTGTCCCACTCCCACATGTCTGGGCACCTGCCCTGGGCTGAGGCCAGGCTGCTCCAAGGGCCGCATGAGCCCTAATCTGCCACAGAGCAACCCAGGTTAAACACAGCCCATGCACAAAGCCACAGGCTAAATCCTGTGGAATTGTTTTTAATGACTGAATTTAACCATTTTCATAGTTGGTTCCTGGAGGTGTGCCAAGTGCCCGCTTGCCTCTTCTAGACCCACAGCTTCTTGATCCACTTGTGGTTTCCATGTCACTAATGTAGAAACATCATGGACTAGCATCCCCAGTCTTTGCCCTCATCCAGCTGTCGCAGCGCACACTGGGGCCTCCCCCCTGCTGCCCAGGGGGGGRCGGGGTGGGCAGCCTCCTGAAACCCATCTTTCTGTGACTGTCTTAGGTGACGTGTAGCCCTCTTCCGTTTTTTCACCCAACAACTTCCTCTGTCCTGCTGCACGGTCCAGAGTCTGGGACCGACTTTGTTTCTTTGTTATTTATGATCTTGTTTAAAGAAAATAAATATCTCCCAACCTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAA

[0121] TABLE 6 S. cerevisiae PME-1 Coding Sequence (SEQ ID NO:9)ATGTCTGACGATTTGAGAAGAAAAATTGCTTTATCCCAGTTTGAGAGAGCCAAGAATGTTCTAGATGCGACATTCCAAGAAGCATACGAGGATGATGAAAACGATGGTGATGCATTAGGTTCCCTGCCATCATTTAATGGACAATCAAATAGGAACAGAAAATATACGGGCAAAACCGGTAGTACTACTGATAGAATTTCAAGTAAGGAAAAGAGTAGTTTACCCACTTGGAGTGATTTTTTTGATAATAAGGAGTTGGTAAGTCTTCCTGATAGAGATCTGGACGTAAATACATACTATACATTACCTACTTCATTGTTATCAAATACCACTTCAATTCCCATCTTTATTTTCCACCATGGGGCGGGCTCCTCAGGTTTATCATTTGCAAACTTGGCCAAGGAATTAAATACTAAACTAGAAGGAAGATGCGGATGCTTTGCATTTGATGCTAGGGGGCATGCAGAAACAAAGTTTAAGAAGGCTGATGCGCCTATATGCTTTGACAGGGACTCTTTTATCAAAGATTTTGTAAGCCTGCTAAATTATTGGTTTAAGTCTAAAATAAGCCAAGAGCCACTTCAGAAGGTATCTGTTATACTAATTGGTCATTCCCTTGGTGGAAGTATATGTACTTTTGCGTACCCTAAATTATCAACAGAACTACAAAAGAAAATTCTTGGTATTACTATGTTAGATATTGTAGAAGAGGCTGCCATTATGGCCTTAAATAAAGTTGAACATTTTTTGCAGAATACACCCAATGTATTTGAATCAATTAATGACGCTGTCGATTGGCACGTTCAACACGCGTTATCGAGATTGAGGTCAAGCGCCGAAATTGCTATACCAGCTTTATTTGCTCCGCTCAAGTCAGGGAAAGTTGTCAGGATAACAAACCTTAAGACCTTTAGCCCTTTCTGGGACACATGGTTTACCGATCTGTCGCACTCCTTTGTTGGCTTACCTGTTAGTAAATTATTAATATTGGCGGGAAACGAAAATCTCGATAAAGAATTAATTGTGGGGCAAATGCAAGGTAAATATCAGTTGGTAGTTTTCCAAGATTCCGGGCATTTCATTCAAGAAGATTCGCCTATAAAAACAGCAATCACTTTAATTGATTTCTGGAAGCGGAACGATTCTAGGAATGTAGTAATCAAGACTAATTGGGGTCAACACAAAACCGTGCAAAATACA TAA

[0122] TABLE 7 C. elegans PME-1 Coding Sequence (SEQ ID NO:1O)ATGTCCGACGATAAATTAGACACTCTTCCGGATCTTCAATCGGAAACGTCACATGTCACAACTCCTCACAGGCAAAATGATCTTCTTCGTCAAGCGGTCACTCATGGAAGGCCACCACCAGTTCCGAGCACATCAACTTCTGGAAAGAAACGAGAAATGTCTGAACTACCGTGGTCAGATTTTTTTGATGAAAAGAAGGACGCAAACATTGATGGAGATGTTTTCAATGTGTACATAAAGGGAAATGAAGGTCCAATTTTCTATTTGCTTCACGGTGGAGGTTATTCAGGCCTCACATGGGCGTGTTTTGCGAAAGAATTGGCAACTTTAATATCATGCAGAGTTGTTGCACCTGATTTAAGAGGACACGGCGACACTAAATGTTCTGATGAGCACGATCTTTCGAAAGAAACCCAAATAAAGGATATTGGAGCAATCTTCAAGAACATTTTCGGCGAAGACGATTCACCAGTATGCATTGTTGGACACAGTATGGGTGGTGCATTGGCCATTCATACATTGAATGCAAAGATGATTTCTTCAAAAGTCGCTGCACTCATTGTCATTGATGTTGTCGAAGGTTCCGCTATGGAAGCACTTGGAGGAATGGTTCATTTTTTACATTCAAGGCCTTCTTCATTTCCTTCTATCGAAAAAGCCATTCACTGGTGCCTTTCTTCGGGTACAGCGAGGAATCCCACAGCTGCACGGGTCTCAATGCCGTCTCAAATTAGAGAAGTATCGGAACACGAGTACACTTGGCGAATTGATTTAACAACAACAGAACAGTACTGGAAAGGATGGTTTGAAGGATTATCCAAAGAATTTTTGGGATGTTCCGTTCCGAAGATGCTTGTTCTAGCGGGCGTTGATCGGCTGGACAGGGATCTCACAATTGGTCAAATGCAGGGAAAGTTTCAGACTTGTGTGTTACCAAAAGTTGGACATTGTGTTCAGGAAGATAGCCCACAAAATCTTGCAGATGAAGTCGGAAGATTCGCTTGCCGCCATAGAATTGCCCAACCGAAATTCTCAGCCCTTGCATCACCACCAGATCCAGCGATTCTCGAATACAGAAAACGTCATCACCAATAA

[0123] TABLE 8 Comparison of the sequences surrounding the putative orknown active site serines of PME-1 proteins and CheB Species Firstresidue Sequence SEQ ID NO: Human PME-1 150 IMLIGHSMG 11 C. elegansPME-1 158 VCIVGHSMG 12 S. cerevisiae PME-1 199 VILIGHSLG 13 S.typhimurium CheB 158 LIAIGASTG 14

[0124]

1 17 1 20 DNA Artificial Sequence Description of Artificial Sequenceoligonucleotide 1 tgttgaggag gggtggacag 20 2 20 DNA Artificial SequenceDescription of Artificial Sequence oligonucleotide 2 tgtatggggaccttcctcct 20 3 16 PRT Homo sapiens 3 Arg Ile Glu Leu Ala Lys Thr GluLys Tyr Trp Asp Gly Trp Phe Arg 1 5 10 15 4 2484 DNA Homo sapiens CDS(100)..(1257) 4 gggcgtcgtt aggggagcga gtcgtgaccg gttgggccac actcaacgtgggacgaagct 60 tcgcctactg tttgactacg tgcgtgcagc ctcccctcg atg tcg gcc ctcgaa 114 Met Ser Ala Leu Glu 1 5 aag agc atg cac ctc ggc cgc ctt ccc tctcgc cca cct cta ccc ggc 162 Lys Ser Met His Leu Gly Arg Leu Pro Ser ArgPro Pro Leu Pro Gly 10 15 20 agc ggg ggc agt cag agc gga gcc aag atg cgaatg ggc cct gga aga 210 Ser Gly Gly Ser Gln Ser Gly Ala Lys Met Arg MetGly Pro Gly Arg 25 30 35 aag cgg gac ttt tcc cct gtt cct tgg agt cag tatttt gag tcc atg 258 Lys Arg Asp Phe Ser Pro Val Pro Trp Ser Gln Tyr PheGlu Ser Met 40 45 50 gaa gat gta gaa gta gag aat gaa act ggc aag gat actttt cga gtc 306 Glu Asp Val Glu Val Glu Asn Glu Thr Gly Lys Asp Thr PheArg Val 55 60 65 tac aag agt ggt tca gag ggt cca gtc ctg ctc ctt ctg catgga gga 354 Tyr Lys Ser Gly Ser Glu Gly Pro Val Leu Leu Leu Leu His GlyGly 70 75 80 85 ggt cat tct gcc ctt tct tgg gct gtg ttc acg gca gcg attatt agt 402 Gly His Ser Ala Leu Ser Trp Ala Val Phe Thr Ala Ala Ile IleSer 90 95 100 aga gtt cag tgt agg att gta gct ttg gat ctg cga agt catggt gaa 450 Arg Val Gln Cys Arg Ile Val Ala Leu Asp Leu Arg Ser His GlyGlu 105 110 115 aca aag gtc aag aat cct gaa gat ctg tct gca gaa aca atggca aaa 498 Thr Lys Val Lys Asn Pro Glu Asp Leu Ser Ala Glu Thr Met AlaLys 120 125 130 gac gtt ggc aat gtg gtt gaa gcc atg tat ggg gac ctt cctcct cca 546 Asp Val Gly Asn Val Val Glu Ala Met Tyr Gly Asp Leu Pro ProPro 135 140 145 att atg ctg att gga cat agc atg ggt ggt gct att gca gtccac aca 594 Ile Met Leu Ile Gly His Ser Met Gly Gly Ala Ile Ala Val HisThr 150 155 160 165 gca tca tcc aac ctg gta cca agc ctc ttg ggt ctg tgcatg att gat 642 Ala Ser Ser Asn Leu Val Pro Ser Leu Leu Gly Leu Cys MetIle Asp 170 175 180 gtt gta gaa ggt aca gct atg gat gca ctt aat agc atgcag aat ttc 690 Val Val Glu Gly Thr Ala Met Asp Ala Leu Asn Ser Met GlnAsn Phe 185 190 195 tta cgg ggt cgt cct aaa acc ttc aag tct ctg gag aatgct att gaa 738 Leu Arg Gly Arg Pro Lys Thr Phe Lys Ser Leu Glu Asn AlaIle Glu 200 205 210 tgg agt gtg aag agt ggc cag att cga aat ctg gag tctgcc cgt gtc 786 Trp Ser Val Lys Ser Gly Gln Ile Arg Asn Leu Glu Ser AlaArg Val 215 220 225 tca atg gtt ggc caa gtc aaa cag tgt gaa gga att acaagt cca gaa 834 Ser Met Val Gly Gln Val Lys Gln Cys Glu Gly Ile Thr SerPro Glu 230 235 240 245 ggc tca aaa tct ata gtg gaa gga atc ata gag gaagaa gaa gaa gat 882 Gly Ser Lys Ser Ile Val Glu Gly Ile Ile Glu Glu GluGlu Glu Asp 250 255 260 gag gaa gga agt gag tct ata agc aag agg aaa aaggaa gat gac atg 930 Glu Glu Gly Ser Glu Ser Ile Ser Lys Arg Lys Lys GluAsp Asp Met 265 270 275 gag acc aag aaa gac cat cca tac acc tgg aga attgaa ctg gca aaa 978 Glu Thr Lys Lys Asp His Pro Tyr Thr Trp Arg Ile GluLeu Ala Lys 280 285 290 aca gaa aaa tac tgg gac ggc tgg ttc cga ggc ttatcc aat ctc ttt 1026 Thr Glu Lys Tyr Trp Asp Gly Trp Phe Arg Gly Leu SerAsn Leu Phe 295 300 305 ctt agt tgt ccc att cct aaa ttg ctg ctc ttg gctggt gtt gat aga 1074 Leu Ser Cys Pro Ile Pro Lys Leu Leu Leu Leu Ala GlyVal Asp Arg 310 315 320 325 ttg gat aaa gat ctg acc att ggc cag atg caaggg aag ttc cag atg 1122 Leu Asp Lys Asp Leu Thr Ile Gly Gln Met Gln GlyLys Phe Gln Met 330 335 340 cag gtc cta ccc cag tgt ggc cat gca gtc catgag gat gcc cct gac 1170 Gln Val Leu Pro Gln Cys Gly His Ala Val His GluAsp Ala Pro Asp 345 350 355 aag gta gct gaa gct gtt gcc act ttc ctg atccgg cac agg ttt gca 1218 Lys Val Ala Glu Ala Val Ala Thr Phe Leu Ile ArgHis Arg Phe Ala 360 365 370 gaa ccc atc ggt gga ttc cag tgt gtg ttt cctggc tgt tagtgacctg 1267 Glu Pro Ile Gly Gly Phe Gln Cys Val Phe Pro GlyCys 375 380 385 ctgtccaccc ctcctcaaca tcgagctctg ttgtaaatac gtcgcaccagaggccactgt 1327 gatgccactg tctcctctcc atcccgccca gccatgtgac actggctcccggtagacggg 1387 caccccgaga tgtaccaacc ttttcatgta ttctgccaaa agcattgttttccagggccc 1447 ttgaccaaca tcggcttccc cagtccaggg ctcccctgct cctttcccttccctgtactg 1507 gggtagctcc tgcctgctct ccctgcgttg cctagggtaa agcctccagatttgccatac 1567 tgagcccctc ttcctagcat caggcgatac atctgagttc aaatgtcttcccaggctcag 1627 ggacctccat tccttgagat tgtcttggca tggcccagcc ctgcctcatgggatggacaa 1687 tgcatggggt ggtctttatt tttccctttc aaataaaaca ctagtcaggtaccgttttat 1747 cccagtcgta ctcttccagg tttggaagac ccagagaggc caagatcccatccttagcca 1807 tagcgagcgg tggtggtgga tagcatcaca agaaacgagc ctgaaaatcaggtccagccg 1867 gtccaagcac atggcctccc atctgggaga gcccactgtc ccactcccacatgtctgggc 1927 acctgccctg ggctgaggcc aggctgctcc aggggcctcc tgcgccctcacctgccacag 1987 agcaacccag gttaaataca gcccatgcac aaagccacag gccaaagcctatggaattgt 2047 ttttaatcat caaatttaac cattttcata actggttcct ggaggtgtgcagtgccccct 2107 tgcctcttca aacctacagc ttctctttgc catttgtgga tttcacatcactccacacag 2167 aaacattaca gcctggcatc cccagtcttt gccttcttcc agctgcctcgacacagcact 2227 gtggcctgtc cctattgccc aggcacgcca tttccaaggg caggaaggggcagtgtcctg 2287 aagcccatct tttctgtgac tgtcttaggt gatgtgtagc cccctccacctttccactca 2347 acaacctccc acccctgtcc tgctgcatgg tccggagtct gggacctactttgttttttg 2407 ttatttatga ccttgtttaa agaaaataaa tatctcccaa cctttaaaaaaaaaaaaaaa 2467 aaaaaaaaaa aaaaaaa 2484 5 386 PRT Homo sapiens 5 Met SerAla Leu Glu Lys Ser Met His Leu Gly Arg Leu Pro Ser Arg 1 5 10 15 ProPro Leu Pro Gly Ser Gly Gly Ser Gln Ser Gly Ala Lys Met Arg 20 25 30 MetGly Pro Gly Arg Lys Arg Asp Phe Ser Pro Val Pro Trp Ser Gln 35 40 45 TyrPhe Glu Ser Met Glu Asp Val Glu Val Glu Asn Glu Thr Gly Lys 50 55 60 AspThr Phe Arg Val Tyr Lys Ser Gly Ser Glu Gly Pro Val Leu Leu 65 70 75 80Leu Leu His Gly Gly Gly His Ser Ala Leu Ser Trp Ala Val Phe Thr 85 90 95Ala Ala Ile Ile Ser Arg Val Gln Cys Arg Ile Val Ala Leu Asp Leu 100 105110 Arg Ser His Gly Glu Thr Lys Val Lys Asn Pro Glu Asp Leu Ser Ala 115120 125 Glu Thr Met Ala Lys Asp Val Gly Asn Val Val Glu Ala Met Tyr Gly130 135 140 Asp Leu Pro Pro Pro Ile Met Leu Ile Gly His Ser Met Gly GlyAla 145 150 155 160 Ile Ala Val His Thr Ala Ser Ser Asn Leu Val Pro SerLeu Leu Gly 165 170 175 Leu Cys Met Ile Asp Val Val Glu Gly Thr Ala MetAsp Ala Leu Asn 180 185 190 Ser Met Gln Asn Phe Leu Arg Gly Arg Pro LysThr Phe Lys Ser Leu 195 200 205 Glu Asn Ala Ile Glu Trp Ser Val Lys SerGly Gln Ile Arg Asn Leu 210 215 220 Glu Ser Ala Arg Val Ser Met Val GlyGln Val Lys Gln Cys Glu Gly 225 230 235 240 Ile Thr Ser Pro Glu Gly SerLys Ser Ile Val Glu Gly Ile Ile Glu 245 250 255 Glu Glu Glu Glu Asp GluGlu Gly Ser Glu Ser Ile Ser Lys Arg Lys 260 265 270 Lys Glu Asp Asp MetGlu Thr Lys Lys Asp His Pro Tyr Thr Trp Arg 275 280 285 Ile Glu Leu AlaLys Thr Glu Lys Tyr Trp Asp Gly Trp Phe Arg Gly 290 295 300 Leu Ser AsnLeu Phe Leu Ser Cys Pro Ile Pro Lys Leu Leu Leu Leu 305 310 315 320 AlaGly Val Asp Arg Leu Asp Lys Asp Leu Thr Ile Gly Gln Met Gln 325 330 335Gly Lys Phe Gln Met Gln Val Leu Pro Gln Cys Gly His Ala Val His 340 345350 Glu Asp Ala Pro Asp Lys Val Ala Glu Ala Val Ala Thr Phe Leu Ile 355360 365 Arg His Arg Phe Ala Glu Pro Ile Gly Gly Phe Gln Cys Val Phe Pro370 375 380 Gly Cys 385 6 400 PRT Saccharomyces cerevisiae 6 Met Ser AspAsp Leu Arg Arg Lys Ile Ala Leu Ser Gln Phe Glu Arg 1 5 10 15 Ala LysAsn Val Leu Asp Ala Thr Phe Gln Glu Ala Tyr Glu Asp Asp 20 25 30 Glu AsnAsp Gly Asp Ala Leu Gly Ser Leu Pro Ser Phe Asn Gly Gln 35 40 45 Ser AsnArg Asn Arg Lys Tyr Thr Gly Lys Thr Gly Ser Thr Thr Asp 50 55 60 Arg IleSer Ser Lys Glu Lys Ser Ser Leu Pro Thr Trp Ser Asp Phe 65 70 75 80 PheAsp Asn Lys Glu Leu Val Ser Leu Pro Asp Arg Asp Leu Asp Val 85 90 95 AsnThr Tyr Tyr Thr Leu Pro Thr Ser Leu Leu Ser Asn Thr Thr Ser 100 105 110Ile Pro Ile Phe Ile Phe His His Gly Ala Gly Ser Ser Gly Leu Ser 115 120125 Phe Ala Asn Leu Ala Lys Glu Leu Asn Thr Lys Leu Glu Gly Arg Cys 130135 140 Gly Cys Phe Ala Phe Asp Ala Arg Gly His Ala Glu Thr Lys Phe Lys145 150 155 160 Lys Ala Asp Ala Pro Ile Cys Phe Asp Arg Asp Ser Phe IleLys Asp 165 170 175 Phe Val Ser Leu Leu Asn Tyr Trp Phe Lys Ser Lys IleSer Gln Glu 180 185 190 Pro Leu Gln Lys Val Ser Val Ile Leu Ile Gly HisSer Leu Gly Gly 195 200 205 Ser Ile Cys Thr Phe Ala Tyr Pro Lys Leu SerThr Glu Leu Gln Lys 210 215 220 Lys Ile Leu Gly Ile Thr Met Leu Asp IleVal Glu Glu Ala Ala Ile 225 230 235 240 Met Ala Leu Asn Lys Val Glu HisPhe Leu Gln Asn Thr Pro Asn Val 245 250 255 Phe Glu Ser Ile Asn Asp AlaVal Asp Trp His Val Gln His Ala Leu 260 265 270 Ser Arg Leu Arg Ser SerAla Glu Ile Ala Ile Pro Ala Leu Phe Ala 275 280 285 Pro Leu Lys Ser GlyLys Val Val Arg Ile Thr Asn Leu Lys Thr Phe 290 295 300 Ser Pro Phe TrpAsp Thr Trp Phe Thr Asp Leu Ser His Ser Phe Val 305 310 315 320 Gly LeuPro Val Ser Lys Leu Leu Ile Leu Ala Gly Asn Glu Asn Leu 325 330 335 AspLys Glu Leu Ile Val Gly Gln Met Gln Gly Lys Tyr Gln Leu Val 340 345 350Val Phe Gln Asp Ser Gly His Phe Ile Gln Glu Asp Ser Pro Ile Lys 355 360365 Thr Ala Ile Thr Leu Ile Asp Phe Trp Lys Arg Asn Asp Ser Arg Asn 370375 380 Val Val Ile Lys Thr Asn Trp Gly Gln His Lys Thr Val Gln Asn Thr385 390 395 400 7 364 PRT Caenorhabditis elegans 7 Met Ser Asp Asp LysLeu Asp Thr Leu Pro Asp Leu Gln Ser Glu Thr 1 5 10 15 Ser His Val ThrThr Pro His Arg Gln Asn Asp Leu Leu Arg Gln Ala 20 25 30 Val Thr His GlyArg Pro Pro Pro Val Pro Ser Thr Ser Thr Ser Gly 35 40 45 Lys Lys Arg GluMet Ser Glu Leu Pro Trp Ser Asp Phe Phe Asp Glu 50 55 60 Lys Lys Asp AlaAsn Ile Asp Gly Asp Val Phe Asn Val Tyr Ile Lys 65 70 75 80 Gly Asn GluGly Pro Ile Phe Tyr Leu Leu His Gly Gly Gly Tyr Ser 85 90 95 Gly Leu ThrTrp Ala Cys Phe Ala Lys Glu Leu Ala Thr Leu Ile Ser 100 105 110 Cys ArgVal Val Ala Pro Asp Leu Arg Gly His Gly Asp Thr Lys Cys 115 120 125 SerAsp Glu His Asp Leu Ser Lys Glu Thr Gln Ile Lys Asp Ile Gly 130 135 140Ala Ile Phe Lys Asn Ile Phe Gly Glu Asp Asp Ser Pro Val Cys Ile 145 150155 160 Val Gly His Ser Met Gly Gly Ala Leu Ala Ile His Thr Leu Asn Ala165 170 175 Lys Met Ile Ser Ser Lys Val Ala Ala Leu Ile Val Ile Asp ValVal 180 185 190 Glu Gly Ser Ala Met Glu Ala Leu Gly Gly Met Val His PheLeu His 195 200 205 Ser Arg Pro Ser Ser Phe Pro Ser Ile Glu Lys Ala IleHis Trp Cys 210 215 220 Leu Ser Ser Gly Thr Ala Arg Asn Pro Thr Ala AlaArg Val Ser Met 225 230 235 240 Pro Ser Gln Ile Arg Glu Val Ser Glu HisGlu Tyr Thr Trp Arg Ile 245 250 255 Asp Leu Thr Thr Thr Glu Gln Tyr TrpLys Gly Trp Phe Glu Gly Leu 260 265 270 Ser Lys Glu Phe Leu Gly Cys SerVal Pro Lys Met Leu Val Leu Ala 275 280 285 Gly Val Asp Arg Leu Asp ArgAsp Leu Thr Ile Gly Gln Met Gln Gly 290 295 300 Lys Phe Gln Thr Cys ValLeu Pro Lys Val Gly His Cys Val Gln Glu 305 310 315 320 Asp Ser Pro GlnAsn Leu Ala Asp Glu Val Gly Arg Phe Ala Cys Arg 325 330 335 His Arg IleAla Gln Pro Lys Phe Ser Ala Leu Ala Ser Pro Pro Asp 340 345 350 Pro AlaIle Leu Glu Tyr Arg Lys Arg His His Gln 355 360 8 2409 DNA Mus musculusmisc_feature (1)..(2409) N is A, T, G or C. 8 ttgtactgca cgtatcgtgggacggacctt gggccactgt tgtcgacgtg cggcctccct 60 ttgatgtcgg cccttgaaaaaagcatgcac ctcggccgcc taccttctcg ccctcctcta 120 cccggcagcg ggggcagtcagagcggacgc aagatgcgga tgggccctgg acggaagcgg 180 gactttaccc ctgtcccatggagtcagtac tttgagtcaa tggaagatgt ggaagtggag 240 aatgaaactg gcaaggatacttttcgagtt tacaagattg gttnnnnnnn nnnnnnnnnn 300 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 360 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 420 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 480 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 540 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 600 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 660 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 720 nnnnnnnnnn nnnnnnnnnnnnnnnnnntt cggatccttg gccaagtcaa acagtgtgaa 780 ggaattacaa gtccagaaggttccaaatcc atagtggaag gaatcataga ggaggaggaa 840 gaagatgagg aaggaagtgagtcagttaac aagaggaaaa aggaagacga catggaaacc 900 aagaaggatc acccatacacctggagaatt gagctggcaa aaacagaaaa gtactgggat 960 ggctggttcc ggggcttatccaatctcttt cttagctgtc ctattcctaa actgctgctc 1020 ttggcgggtg ttgacagattggataaagat ctgaccatag gccagatgca ggggaagttc 1080 cagatgcagg tcttaccccagtgtggccat gcagtccatg aggatgcccc tgacaaggta 1140 gctgaagctg ttgccactttcctgatccgg cacaggtttg cagagcccat cggaggattc 1200 cagtgtgtgt ttactggctgctagtgacct gctgtctact cctccctcta cattgagctc 1260 tgttgtaaat acatcgcaccagaggccact gtgacgccgc tgtctcctcc tctccatccc 1320 gcccagccat gtgacaccggctcttgtaga gggcatcccc agatgtccaa accctttcct 1380 gtgtactgtt gaaagcattgttcttcaggg cccttgtcca acagtggccc gtgcagtctg 1440 gggtccacag ctcttcctctccttcctgtg ctccctgcct tgcctaggat gaagcctcca 1500 gcgctgctcc ctggccctgttcctggcata tggcaatgta ccccaggctc agggatctcc 1560 cttccttgag gatgttcttggcatggtcct gccctacctc atgggatggg caatgcacac 1620 actggccctt atttttccctttcaaataaa acaccagtca ggtaccttta tcccagtctt 1680 aactgtccca aatctggaaggtccagagta agcaggattc agggagaggg agtggatagc 1740 aagtatccca agaaaccaacctgtaagtca ggtccagcca gtccaagcac atggcttccc 1800 atctgggtga gcccactgtcccactcccac atgtctgggc acctgccctg ggctgaggcc 1860 aggctgctcc aagggccgcatgagccctaa tctgccacag agcaacccag gttaaacaca 1920 gcccatgcac aaagccacaggctaaatcct gtggaattgt ttttaatgac tgaatttaac 1980 cattttcata gttggttcctggaggtgtgc caagtgcccg cttgcctctt ctagacccac 2040 agcttcttga tccacttgtggtttccatgt cactaatgta gaaacatcat ggactagcat 2100 ccccagtctt tgccctcatccagctgtcgc agcgcacact ggggcctccc cctgctgccc 2160 agggggggrc ggggtgggcagcctcctgaa acccatcttt ctgtgactgt cttaggtgac 2220 gtgtagccct cttccgttttttcacccaac aacttcctct gtcctgctgc acggtccaga 2280 gtctgggacc gactttgtttctttgttatt tatgatcttg tttaaagaaa ataaatatct 2340 cccaaccttt aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2400 aaaaaaaaa 2409 9 1202DNA Saccharomyces cerevisiae 9 tgtctgacga tttgagaaga aaaattgctttatcccagtt tgagagagcc aagaatgttc 60 tagatgcgac attccaagaa gcatacgaggatgatgaaaa cgatggtgat gcattaggtt 120 ccctgccatc atttaatgga caatcaaataggaacagaaa atatacgggc aaaaccggta 180 gtactactga tagaatttca agtaaggaaaagagtagttt acccacttgg agtgattttt 240 ttgataataa ggagttggta agtcttcctgatagagatct ggacgtaaat acatactata 300 cattacctac ttcattgtta tcaaataccacttcaattcc catctttatt ttccaccatg 360 gggcgggctc ctcaggttta tcatttgcaaacttggccaa ggaattaaat actaaactag 420 aaggaagatg cggatgcttt gcatttgatgctagggggca tgcagaaaca aagtttaaga 480 aggctgatgc gcctatatgc tttgacagggactcttttat caaagatttt gtaagcctgc 540 taaattattg gtttaagtct aaaataagccaagagccact tcagaaggta tctgttatac 600 taattggtca ttcccttggt ggaagtatatgtacttttgc gtaccctaaa ttatcaacag 660 aactacaaaa gaaaattctt ggtattactatgttagatat tgtagaagag gctgccatta 720 tggccttaaa taaagttgaa cattttttgcagaatacacc caatgtattt gaatcaatta 780 atgacgctgt cgattggcac gttcaacacgcgttatcgag attgaggtca agcgccgaaa 840 ttgctatacc agctttattt gctccgctcaagtcagggaa agttgtcagg ataacaaacc 900 ttaagacctt tagccctttc tgggacacatggtttaccga tctgtcgcac tcctttgttg 960 gcttacctgt tagtaaatta ttaatattggcgggaaacga aaatctcgat aaagaattaa 1020 ttgtggggca aatgcaaggt aaatatcagttggtagtttt ccaagattcc gggcatttca 1080 ttcaagaaga ttcgcctata aaaacagcaatcactttaat tgatttctgg aagcggaacg 1140 attctaggaa tgtagtaatc aagactaattggggtcaaca caaaaccgtg caaaatacat 1200 aa 1202 10 1095 DNA Caenorhabditiselegans 10 atgtccgacg ataaattaga cactcttccg gatcttcaat cggaaacgtcacatgtcaca 60 actcctcaca ggcaaaatga tcttcttcgt caagcggtca ctcatggaaggccaccacca 120 gttccgagca catcaacttc tggaaagaaa cgagaaatgt ctgaactaccgtggtcagat 180 ttttttgatg aaaagaagga cgcaaacatt gatggagatg ttttcaatgtgtacataaag 240 ggaaatgaag gtccaatttt ctatttgctt cacggtggag gttattcaggcctcacatgg 300 gcgtgttttg cgaaagaatt ggcaacttta atatcatgca gagttgttgcacctgattta 360 agaggacacg gcgacactaa atgttctgat gagcacgatc tttcgaaagaaacccaaata 420 aacgatattg gagcaatctt caagaacatt ttcggcgaag acgattcaccagtatgcatt 480 gttggacaca gtatgggtgg tgcattggcc attcatacat tgaatgcaaagatgatttct 540 tcaaaagtcg ctgcactcat tgtcattgat gttgtcgaag gttccgctatggaagcactt 600 ggaggaatgg ttcatttttt acattcaagg ccttcttcat ttccttctatcgaaaaagcc 660 attcactggt gcctttcttc gggtacagcg aggaatccca cagctgcacgggtctcaatg 720 ccgtctcaaa ttagagaagt atcggaacac gagtacactt ggcgaattgatttaacaaca 780 acagaacagt actggaaagg atggtttgaa ggattatcca aagaatttttgggatgttcc 840 gttccgaaga tgcttgttct agcgggcgtt gatcggctgg acagggatctcacaattggt 900 caaatgcagg gaaagtttca gacttgtgtg ttaccaaaag ttggacattgtgttcaggaa 960 gatagcccac aaaatcttgc agatgaagtc ggaagattcg cttgccgccatagaattgcc 1020 caaccgaaat tctcagccct tgcatcacca ccagatccag cgattctcgaatacagaaaa 1080 cgtcatcacc aataa 1095 11 9 PRT Homo sapiens 11 Ile MetLeu Ile Gly His Ser Met Gly 1 5 12 9 PRT Caenorhabditis elegans 12 ValCys Ile Val Gly His Ser Met Gly 1 5 13 9 PRT Saccharomyces cerevisiae 13Val Ile Leu Ile Gly His Ser Leu Gly 1 5 14 9 PRT Salmonella typhimurium14 Leu Ile Ala Ile Gly Ala Ser Thr Gly 1 5 15 10 PRT Artificial sequenceUNSURE (1) X at position 1 is Leu, Ile or Val. 15 Xaa Xaa Xaa Xaa GlyXaa Ser Xaa Gly Xaa 1 5 10 16 51 DNA Homo sapiens 16 tgactacgtgcgtgcagcct cccctcgatg tcggccctcg aaaagagcat g 51 17 7 PRT ArtificialSequence Description of Artificial Sequence Sequence resulting fromalternative splicing. 17 Leu Leu Ser Thr Tyr Cys Arg 1 5

We claim:
 1. An isolated nucleic acid molecule comprising a portion encoding protein phosphatase methylesterase-1 (PME-1), wherein said portion comprises a sequence at least 70% identical to a nucleotide sequence given in SEQ ID NO:9, nucleotides 1-1200.
 2. The nucleic acid molecule of claim 1, wherein said molecule encodes a PME-1 polypeptide consisting essentially of an amino acid sequence as given in SEQ ID NO:6, amino acids 1-400.
 3. The nucleic acid molecule of claim 2, wherein said molecule comprises a PME-1 coding sequence as shown in SEQ ID NO:9, nucleotide 1-1200.
 4. A recombinant expression vector comprising the nucleic acid molecule encoding protein phosphatase methylesterase-1 (PME-1) of claim 1, wherein a coding sequence of said molecule is operably linked to and expressed under control of transcription and translation regulatory elements.
 5. The expression vector of claim 4, wherein the encoded PME-1 consists essentially of the amino acid sequence given in SEQ ID NO:6, amino acids 1-400.
 6. The expression vector of claim 4, wherein said vector comprises the PME-1 coding sequence substantially as given in SEQ ID NO:9, nucleotides 1-1200.
 7. The expression vector of claim 4, wherein said vector is a bacterial vector.
 8. The expression vector of claim 4, wherein said vector is a baculovirus vector.
 9. The expression vector of claim 4, wherein said vector is a mammalian vector.
 10. A recombinant host cell, wherein said cell comprises the expression vector of claim
 4. 11. The host cell of claim 10, wherein said cell is a recombinant bacterial cell.
 12. The host cell of claim 10, where said cell is a recombinant mammalian cell.
 13. A method for producing a recombinant protein phosphatase methylesterase-1 (PME-1) polypeptide, said method comprising the steps of: (a) introducing the expression vector of claim 4 into a host selected from the group consisting of bacteria, insects and mammals; and (b) culturing under conditions where PME-1 polypeptide is produced, whereby said PME-1 polypeptide shows protein phosphatase methylsterase activity in vitro.
 14. An isolated polypeptide or fragment thereof, having protein phosphatase methylesterase-1 activity, the amino acid sequence of which comprises residues 1-400 of SEQ ID NO:
 6. 